LIBRARY OF CONGRESS. 

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Slielf...V!:;i.. 



UNITED STATES OF AMERICA. 






^It^^i/. I ;,:•'-.-■. 














MEDICAL MICROSCOPY. 



WETHERED. 



JUST READY. FOURTH EDITION. 



Swanzy. Diseases of the Eye. 

Enlarged and Improved. 176 Illustrations. 

Diseases of the Eye and their Treatment. A Hand- 
book for Physicians and Students. By Henry R. 
Swanzy, a.m., m.b., f.r.c.s.i.. Surgeon to the National 
Eye and Ear Infirmary ; Ophthalmic Surgeon to the 
Adelaide Hospital, Dublin ; Examiner in Ophthalmic 
Surgery in the Royal University of Ireland. Fourth 
Edition, Thoroughly Revised. 176 Illustrations. i2mo. 

Cloth, $3.00; Sheep, ^3.50 

" On looking this little volume over carefully, one cannot avoid being 
impressed by the great and well-arranged mass of information presented : 
a good, closely written chapter on elementary optics ; the light, color, and 
form sense ; color blindness and the field of vision. The chapters on 
refraction and accommodation and the ophthalmoscope are models of clear- 
ness and conciseness." — Gaillard' s Medical Jour 7ial, Nezv York. 

Philadelphia : P. Blakiston, Son & Co. 



MEDICAL MICROSCOPY 



A GUIDE TO THE USE OF THE MICROSCOPE 
IN MEDICAL PRACTICE 



FRANK J. WETHERED, M.D. (Lond.) 

MEMBER OF THE ROYAL COLLEGE OF PHYSICIANS ; MEDICAL REGISTRAR TO THE 

MIDDLESEX HOSPITAL AND DEMONSTRATOR OF PRACTICAL MEDICINE 

TO THE MIDDLESEX HOSPITAL MEDICAL SCHOOL; LATE 

ASSISTANT PHYSICIAN TO THE CITY OF LONDON 

HOSPITAL FOR DISEASES OF THE CHEST, 

VICTORIA PARK 



WITH ILLUSTRATIONS 



PHILADELPHIA -^1 

BLAKISTON, SON & QOjS^^ 

IOI2 WALNUT STREET ^^ 'K^ 

A 
1892 






^^ 



Copyright, 1892, by P. Blakiston, Son & Co. 



Press of Wm. F. Fell &. Co., 
1220-24 Sansom St., 

PHILADELPHIA. 



d 



RICHARD DOUGLAS POWELL, M.D., F.R.C.P. 

PHYSICIAN EXTRAORDINARY TO HER MAJESTY THE QUEEN; 

CONSULTING PHYSICIAN TO THE HOSPITAL FOR CONSUMPTION AND 

DISEASES OF THE CHEST, BROMPTON ; 

PHYSICIAN TO THE MIDDLESEX HOSPITAL," 

'Qtlis Bock is ^tUtKUl:^ 

WITH THE SINCERE RESPECT AND GRATITUDE OF 

THE AUTHOR. 



PREFACE. 



Within recent years the microscope has gradually been 
brought more and more into use as an aid to diagnosis. 
In certain classes of diseases, notably those of the chest 
and urinary organs, it has become almost a routine 
practice, in hospitals at any rate, to examine the sputa 
and deposits, and the use of the microscope is rapidly 
being extended to private practice. 

In this work an effort has been made to lay before 
practitioners and students the most simple methods of 
preparing microscopic sections, and of the examination 
of urinary deposits, sputa, blood, etc. At the same 
time, for those who wish to extend their studies further, 
more elaborate methods have been detailed, and chap- 
ters added on the examination of food and bacteriological 
methods. It would have been beyond the scope of the 
book to have given descriptions of all the pathological 
changes found in the different organs, but a chapter 
on '^Tumors'* has been inserted, as for purposes of 
diagnosis, such examinations are frequently made in the 
operating theatre or post-mortem room. 

Many of the illustrations are original, but I have also 
to thank Profs. Ziegler, von Jaksch, and Crookshank, 
Drs. Troup, Parkes, and Crocker, for permission to use 
many of their figures. 

I have also to thank Prof. Stirling, Drs. Vincent 
Harris and Sims Woodhead, for allowing me to copy 

vii 



VIU PREFACE. 

many formulae from their works, and especially Dr. 
Wynter for consenting to my making use of the material 
collected for our joint work on '^ Clinical and Practical 
Pathology." Also my thanks are due to Mr. Mansell 
MouUin, Mr. Eve, Dr. A. Garrod, and Dr. Herman, for 
their kind advice and help, also to Mr. Lewis for his 
aid in the matter of illustrations, and to the various 
firms who have provided cliches of apparatus. 

Queen An7ie Street. 



CONTENTS. 



CHAPTER I. 

The Microscope and Accessories. 

PAGE 

Choice of an Instrument — Compound Microscope — Requisites of 
a good microscope : stage, diaphragm — Qualities of a good 
objective : defining power, flatness of field, penetrating 
power, angle of aperture, resolving power — Nose-piece — 
Oculars — Camera-lucida — Makers of microscopes — Use of the 
microscope — xVbbe's Condenser — Microscope lamp — Drawing 
objects — Micrometer — Accessory apparatus — Cathcart's micro- 
tome — Swift's microtome — Williams' freezing microtome — 
Reichert's microtome — Rocking microtome — Valentin's knife 
— Glass slides — Cover-glasses — Normal saline solution — 
Iodine in iodide of potassium (Gram's solution), 9 

CHAPTER II. 

Hardening and Decalcifying. 

General remarks — Choice of hardening fluid — Absolute alcohol — 
Methylated spirit — Miiller's fluid — Bichromate of potash — 
Chromic acid — Chromic acid and spirit — Ammonium chro- 
mate — Bichromate of ammonia — Corrosive sublimate — Plati- 
num chloride — Osmic acid — Picric acid — Decalcifying fluids — 
Nitric acid — Perenzi's solution — Hydrochloric acid — Von 
Ebner's solution — Picric acid, 42 

CHAPTER III. 

Embedding. 

General remarks — Freezing media — Gum — Dextrin — Simple em- 
bedding — Infiltration methods — Embedding in celloidin : and 
in paraffin, 55 

ix 



X CONTENTS. 

CHAPTER IV. 

Cutting Sections. 

PAGE 

General remarks — Cutting with Cathcart's microtome — Cutting 
with Swift's microtome — Use of Williams' ice freezing micro- 
tome — Cutting sections embedded in celloidin — Cutting with 
Reichert's microtome — Collodionization of sections — Cutting 
sections in paraffin — Cutting in series — Cutting with rocking 
microtome, 62 



CHAPTER V. 

Staining. 

General Remarks — Choice of a stain — Staining with haematoxylin 
and eosin — Staining with carmine and picric acid — Lithium 
carmine — Acidulated alcohol — Picro-carmine — Alum-carmine 
— Ammonium carmine — Borax carmine — Staining in bulk — 
Safranin — Iodine green — General stains : Bismarck brown, 
rubin, orseille — Multiple staining — Treble staining, .... 74 



CHAPTER VI. 

Selective Stains. 

General remarks — INIetallic stains : nitrate of silver, chloride of 
gold, osmic acid — Staining micro-organism: methylene blue, 
Loffler's method, Klihne's method, Mast-zellen, Gram's 
method, Weigert's modification of Gram's method, Neelsen- 
Ziehl method — Methods of staining actinomyces : amyloid re- 
action, karyokinetic figures, 92 



CHAPTER VII. 

Clearing and Mounting. 

Fluid media : glycerine, glycerine jelly, glycerine mounting fluid, 
Farrant's solution, iodine mounting fluid — Cementing mate- 
rials — Mounting in solidifying media — Clearing agents : Can- 
ada balsam, dammar varnish — Breaking down old specimens, 1 10 



CONTENTS. XI 



CHAPTER VIII. 



Complete Processes for the Preparation of Sections. 

PAGE 

First method — Second method, 122 



CHAPTER IX. 

Method of Preparing Sections of the Central 
Nervous System. 

Hardening — Cutting — Staining: Weigert's method, Pal's method, 
Pal-Exner method, aniUne blue-black, Jolgi's sublimate 
method, 125 

CHAPTER X. 

Injection of Tissues. 

Cold injection — Carmine gelatine injection mass, 135 

CHAPTER XI. 

Examination of Fresh Tissues. 

Normal saline solution — Serous fluid — Iodized serum — Teasing — 

Softening — Landois' fluid, . 139 

CHAPTER XII. 

Preparation of Individual Tissues and Organs. 

Epithelium — Areolar tissue — White fibrous tissue — Elastic tissue — 
Adenoid tissue — Adipose tissue — Gelatinous or embryonal 
tissue — Cartilage — Bone — Teeth — Muscle — Nervous system — 
Circulating system — Glandular tissue — The eye — Internal ear 
— Digestive system — Lungs — Thyroid gland — Liver, spleen, 
etc. — Genito-urinary system, 144 

CHAPTER XIII. 

The Examination of Tumors. 

Fibromata — Myxomata — Enchondromata — Osteomata — Myofibro- 
mata — Sarcomata — Papillomata — Adenomata — Epitheliomata 
— Carcinomata, 153 



Xll CONTENTS. 

CHAPTER XIV. 
Examination of Urinary Deposits. 

PAGE 

Method of examination — Organized deposits : epithelium, red blood 
corpuscles, leucocytes, mucus, fat, casts, spermatozoa, frag- 
ments of morbid growth — Pathogenic micro-organisms : tuber- 
cle bacilli, erysipelas — Septic micro-organisms — Sarcinae, bil- 
harzia hcematobia, filaria sanguinis hominis, eustrongylus 
gigas, echinococci — Xon-pathogenic micro-organisms — Unor- 
ganized deposits — Sediments occurring in acid urine : uric 
acid, amorphous urates, urate of soda, oxalate of lime, sulphate 
of lime, cystin, xanthin, tyrosin, leucin, hippuric acid, hcema- 
toidin and bilirubin, soaps of lime and magnesia — Sediments 
occurring in alkaline urine : triple phosphates, neutral phos- 
phate of lime, basic magnesium phosphate, amorphous phos- 
phate of lime, carbonate of lime, urate of ammonia, cholesterin, 
indigo — Gravel and calculi — Extraneous matters — Preservation 
of urinary deposits, 167 



CHAPTER XV. 

Examination of the F.^ces. 

Method of examination — Macroscopic examination — Foreign 
bodies — Mucus and fibrinous shreds — Portions of bowel 
separated from intussusceptions — Intestinal concretions — 
Gall-stones — ^Microscopical examination : food matters, crystals, 
red blood corpuscles, leucocytes, epithelium — Vegetable para- 
sites : bacillus of tubercle, bacillus of typhoid fever, bacillus of 
cholera, bacillus of cholera nostras — Xon-pathogenic organ- 
isms — Animal parasites : amoeba coli, infusoria — Taenia medio- 
canellata — Taenia solium — Taenia nana — Bothriocephalus 
latus — Distoma hepaticum — Distoma lanceoktum— Distoma 
sinense — Ascaris lumbricoides — Ascaris mistax — Oxyuris 
vermicularis — Tricocephalus dispar — Anchylostoma duode- 
nale — Strongylus gigas — Anguillula intestinalis — Trichina 
spiralis, 21^ 



CONTENTS. XIU 

CHAPTER XVI. 

Examination of Sputum. 

PAGE 

Method of examination : quantity, consistence, color, odor, bron- 
chial casts — Microscopical examination : leucocytes and mucus 
corpuscles, compound granule cells, red blood cells, epithelium, 
elastic tissue, Curschmann's spirals, tonsillar casts, corpora 
amylacea, portions of membrane, crystals, Charcot- Leyden 
crystals, concretions, foreign bodies, accidental ingredients, . 248 



CHAPTER XVn. 

The Micro-organisms of Sputum. 

Tubercle bacilli : Neelsen-Ziehl method, Ehrlich's method, Gab- 
bett's method, Gibbes' method — Pneumococci — Actinomyces — 
Micrococcus tetragonus — Septic micro-organisms — Fungi — 
Infusoria — Entozoa, 281 



CHAPTER XVIII. 

Examination of Vomit. 

Method of examination — Food matters — Epithelium — Red blood 

cells — Leucocytes — Sarcinse — Yeasts — Micro-organisms, . , 295 



CHAPTER XIX. 

Examination of Discharges and Contents of 
Cavities. 

Serous exudations — Hemorrhagic exudations — Chylous exuda- 
tions — Sero-purulent exudations — Putrid exudations — Puru- 
lent exudations : micro-organisms — Tubercle bacilli — Acti- 
nomyces — Bacillus of leprosy — Bacillus of anthrax — 
Bacillus of syphihs — Gonococcus — Bacillus of Glanders — 
Trichina spiralis — Cysticercus cellulosae — Hydatid cysts — 
Ovarian cysts — Dermoid cysts — Hydro -nephrosis — Sperma- 
tocele, 299 

I 



XIV CONTENTS. 

CHAPTER XX. 

Discharges from the Genital Organs. 

PAGE 

Discharges from the male genital organs : seminal fluid, spermatic 
stains — Discharges from the female genital organs : vaginal 
secretion, Trichomonas vaginalis — Membranes : mucus, 
fibrinous clots, vaginal casts, membranous dysmenorrhoea, 
membranes of early abortion, . . , . 312 



CHAPTER XXI. 

Examination of the Blood. 

Red corpuscles — White corpuscles — Blood plates — Counting the 
corpuscles — Gowers' haemocytometer, Thoma-Zeiss hsemo- 
cytometer — Estimation of the amount of haemoglobin in the 
blood: Gowers' haemoglobinometer, v. Fleischl's haemo- 
meter — Olygocythaemia — Polycythaemia — Leucocythaemia — 
Leucocytosis — Microcythaemia — Poikilocytosis — Melanae- 
mia — Vegetable parasites : bacillus of anthrax, tubercle, 
typhoid fever, glanders and tetanus, spirillum of relapsing 
fever, haematozoa of malaria — Animal parasites : Filaria 
sanguinis hominis, Bilharzie haematobia — Examination of 
blood stains, 321 

CHAPTER XXH. 

Cutaneous Parasites 

Vegetable parasites ; Tricophyton tonsurans, Microsporon furfur, 
Achorion Schonleinii — Animal parasites : pediculi, Sarcoptes 
hominis, Acarus foUiculorum, 348 

CHAPTER XXni. 

Examination of Food and Water. 

Wheat — Barley — Rye — Oatmeal — Maize — Rice — Beans — Peas — 
Sago — Tapioca — Arrowroot — Potato — Turmeric — Bread — 
Flour — Tea — Coffee — Cocoa — Examination of drinking 
water, 354 



CONTENTS. XV 

CHAPTER XXIV. 
Bacteriological Methods. page 

General Considerations — Apparatus required : steam-sterilizer, 
hot-air sterilizer, hot-water filter, incubator, accessories — 
Cultivating media — Solid media : potato cultivations, nutrient 
gelatine, nutrient agar-agar, glycerine agar-agar, sterilized 
blood serum — Liquid media : bouillon, liquid blood serum, 
Pasteur's fluid, Cohn's fluid — Mode of using the solid nutrient 
media : test-tube cultivations, plate cultivations — Mode of using 
the liquid media: drop cultures — Examination of micro- 
organisms : cover-glass preparations, cover-glass impressions — 
Experiments on animals, 369 



LIST OF ILLUSTRATIONS. 



FIG. PAGE 

1. Diagram of Optical Principle of the Microscope, lo 

2. Baker's New Student's Microscope, 13 

3. Beck's Pathological Microscope, 15 

4. Swift's Bacteriological Microscope, , 17 

5. Nose-piece, 18 

6. Bausch and Lamb's Universal Microscope, 21 

7. Abbe Chromatic Condenser, 24 

8. Microscopic Lamp, 26 

9. Prismatic Camera-lucida, 28 

10. Abbe's Camera-lucida, 28 

11. Eye-piece Micrometer, 29 

12. Frazer's Modification of Cathcart's Microtome, 32 

13. Clamp of Cathcart's Microtome, . , ^, 33 

14. WiUiams' Ether Microtome ('* Swift's Microtome"), . ... 34 

15. Williams' Ice and Salt Microtome, 35 

16. Reichert's Microtome, -^7 

17. Rocking Microtome, 38 

18. Valentin's Knife, 39 

19. Diagram for Embedding Box, 57 

20. Cells from a Periosteal Myxoma of the Thigh, 155 

21. Section from an Osteoid Chondroma of the Humerus, ... 156 

22. Section Through the Margin of a Sarcoma Affecting the 

Intermuscular Connective Tissue, . . 158 

23. Section from a Giant-celled Sarcoma, showing also Spindle- 

shaped Cells, 159 

24. Fibro-adenoma of Mamma, 162 

25. Section from an Epithelioma of Skin, 164 

26. Carcinoma of the Mamma, 165 

27. Vaginal Epithelium, 170 

xvii 



XVlll LIST OF ILLUSTRATIONS. 

FIG. PAGE 

28. Epithelium from the Bladder, Ureter, and Pelvis of the Kidney, 1 7 1 

29. Renal Epithelium, Healthy and Fatty, 172 

30. Blood Corpuscles in Urine, 173 

31. Pus-Cells in Urine Affected by Acetic Acid and Unaltered, 177 

32. Urinary Casts, 183 

33. Spermatozoa, 188 

34. Uric Acid, 199 

35. Amorphous Urates, 201 

36. Urate of Soda, 202 

37. Oxalate of Lime, 203 

38. Cystin, 205 

39. Tyrosin, 207 

40. Leucin, 209 

41. Triple Phosphates, 210 

42. Stellar Phosphates, . 211 

43. Cholesterin, 214 

44. Head of Taenia Mediocanellata, 238 

45. Head of Taenia Solium, 240 

46. Ova of Intestinal Parasites, 244 

47. Trichocephalus Dispar, 246 

48. Bronchial Stolon, 256 

49. Various Cells in Sputum, 261 

50. Elastic Tissue, 265 

51. Elastic Tissue Partially Encrusted with Lime Salts, .... 268 

52. Curschmann's Spirals, 269 

53. Cast of a Tonsillar Crypt, 271 

54. Charcot- Leyden Crystals, 274 

55. Silk Fibres, 278 

56. Cotton Fibres, 278 

57. Linen Fibres, • . . . 279 

58. Wool Fibres, 279 

59. Bacterium Pneumonia Crouposa, 289 

60. Oidium Albicans, 292 

61. Vomit, 296 

62. Trichina Spiralis in Muscle, . . 307 

63. Head of Cysticercus Cellulosae, 307 

64. Hydatids, 309 

65. Wall of Hydatid Cyst, 310 



LIST OF ILLUSTRATIONS. XIX 

FIG. PAGE 

66. Normal Semen, 313 

67. Trichomonas Vaginalis, 317 

68. Gower's Heemocytometer, 323 

69. Pipette (Thoma-Zeiss Haemocytometer), 326 

70. Counting Slide (Thoma-Zeiss Apparatus), 327 

71. Cross-section of Thoma-Zeiss Apparatus, 327 

72. Counting Scale (Thoma-Zeiss Apparatus), 328 

73. Gower's Haemoglobinometer, 331 

74. Von Fleischl's Heemometer, 332 

75. Poikilocytosis, 339 

76. Filaria Sanguinis Hominis, 343 

77. Bilharzia Haematobia, 346 

78. Tricophyton Tonsurans, 349 

79. Achorion Schonleinii, 351 

80. Wheat-starch Grains, 355 

81. Barley-starch Grains, 356 

82. Oatmeal-starch Grains, 357 

S^. Maize-starch Grains, 358 

84. Rice-starch Grains, 358 

85. Bean-starch Grains, 359 

86. Pea-starch Grains, , . 359 

87. Sago-starch Grains, 360 

88. Tapioca-starch Grains, 360 

89. Arrowroot-starch Grains, 361 

90. Potato-starch Grains, 361 

91. Acarus Farinae, 363 

92. Coffee, Cells of Testa and Cellular Structure, 365 

93. Chicory, Dotted Ducts and Cellular Structure, 365 

94. Cocoa-starch Cells, 366 

95. Koch's Steam Sterilizer, 371 

96. Hot-water Filtering Apparatus with Ring Burner, .... 372 

97. Babes' Incubator, 374 

98. Platinum Needles, Straight, Hooked, Looped, 375 

99. Method of Inoculating a Test-tube Containing Sterile Nutri- 

ent Jelly, 3S3 

ICXD. Method of Inoculating Test-tubes in the Preparation of 

Plate Cultivations, 385 

loi. Damp Chamber for Plate Cultivations, 387 



MEDICAL MICROSCOPY. 



CHAPTER L 
THE MICROSCOPE AND ACCESSORIES. 

The first question that naturally arises when preparing 
to study the use of the microscope in medicine is as to the 
choice of an instrument. The number of different 
forms of microscopes now in the market is legion, and it 
is an exceedingly difficult matter to make a selection of 
any particular one. In order to do so, an intimate knowl- 
edge of its structure and mode of working is necessary, and 
it is very advisable that an inexperienced person, instead 
of choosing for himself, should seek the advice of a more 
expert friend to help him in his search, for nothing is 
more annoying, after expending a large sum on a large, 
showy stand, than to find that for practical scientific work 
it is almost useless. 

Before stating in detail the requisites of a good micro- 
scope, it will be well to give a short description of the 
instrument as sold by all modern makers. 

Microscopes are distinguished as simple or com- 
pound. In the former, the rays which enter the eye 
of the observer come from an object brought near to it 
after refraction through either a simple lens, or a combi- 
nation of lenses acting as a simple lens, its action as a 
^' magnifier" depending on its enabling the eye to form a 
2 9 



lO 



MEDICAL MICROSCOPY. 



distinct image of the object at a much shorter distance 
than would otherwise be possible. 

The ** compound microscope" consists of at least 
two lenses, so placed relatively to the object, to the eye, 
and to one another, that an enlarged image of the object, 
formed by the lens placed nearest to it (the ^'object- 
glass'^), is looked at through the lens nearest the eye 
(the '* eye-piece "), which acts as a simple microscope in 
magnifying it ; so that the compound microscope may be 
described as a simple microscope applied to look at an 




Diagram of Optical Principle ot the Microscope. 



enlarged image of the object, instead of at the object 
itself. 

This description of the compound microscope will be 
made clearer by referenc to the above diagram (Fig. i). 

^ ^ is the object placed in front of the object-glass L. An 
inverted and somewhat magnified image, db\ is formed on 
the opposite side of the glass. P is the eye-piece. Now 
the distance between the two lenses, L and P, is such that 
the position of the image a!b' is between the eye-piece and 
its focus. From this it follows that for the eye at E^ 



THE MICROSCOPE AND ACCESSORIES. II 

looking through the eye-piece at the image, the same 
effect is produced as with a simple microscope, so that 
instead of the image a!b' , another image AB is seen, 
which is virtual. This second image is also inverted in 
reference to the object. 

The lenses are set in a tube blackened internally, 
the eye-pieces fitting into the upper end, whilst the 
objectives are screwed on to the lower end. This 
tube, which can be lengthened to some extent telescope- 
fashion, is supported on a firm metal *' stand," con- 
sisting of two chief portions, the ** column" and the 
**base." 

The tube is capable of being raised or lowered without 
lateral deviation, this being accomplished by means of a 
telescopic joint, or a rack and pinion arrangement in the 
better class of instruments. These mechanical appliances 
are known as ** adjustments." There are usually two 
such, the ** coarse " and the ** fine." The coarse adjust- 
ment has just been described ; the '"• fine adjustment " con- 
sists of a fine screw usually placed at the top of the column. 

Projecting from the column is a horizontal plate, the 
'' stage," for the reception of the object to be examined. 
It possesses a circular aperture for the passage of light, the 
amount of illumination being determined by *' dia- 
phragms " of different sizes. Below the stage is a 
swinging mirror capable of being moved ia all direc- 
tions, and having two surfaces, a plane and a concave. 
Inserted into the stage are two clips for fixing the glass 
slides in any desired position. 

In microscopes intended for bacteriological work, a 
** sub-stage " is inserted between the mirror and the 
stage proper; this carries the '* condenser '* and its 
diaphragms, and by means of screws can be moved laterally, 
or upward and downward. 



12 MEDICAL MICROSCOPY. 

A second *^ condenser" is usually supplied for purposes 
of looking at opaque objects by reflected light ; this, how- 
ever, is seldom, if ever, required for medical work, all the 
objects being transparent and examined by transmitted light 
obtained from below. 

The column is connected with the base by means of a 
stiff hinge, so that the tube can be fixed either in a per- 
pendicular or horizontal position, or at any angle. 

What, then, are the requisites of a good microscope? 

For useful practical work, it is desirable not to have too 
large an instrument. Especially should a binocular be 
avoided. It is cumbersome, the number of screws around 
the stage becomes bewildering, and no advantage is to be 
gained by it for medical work over a well-made mon- 
ocular, with a simple sub-stage condenser. 

Steadiness is essential. This is secured by a wide base. 
In nearly all microscopes the pedestal is either of the 
horse-shoe, or some modification of the tripod pattern ; it 
should be heavy enough to allow of both adjustments being 
turned, and of the object being freely moved on the stage 
without shaking the instruaient. 

The column must, of course, be perfectly rigid, and 
this property is secured by the use of good, stout metal. 

Both adjustments should turn easily and regularly, 
especially the fine one, for if this be at all stiff, or if the 
thread be .in the least irregular, great annoyance will be 
experienced. 

The stage is usually three inches long by two and a 
half wide. It is not advisable to have a '^movable" 
stage. By means of the fingers the glass slide can be 
moved more readily, and by practice quite as smoothly 
as by any mechanical contrivance, whilst extra screws and 
levers are apt to be in the way. 

There should be plenty of room between the mirror and 



THE MICROSCOPE AND ACCESSORIES. 



^3 



the stage, so as to allow the insertion of a condenser ; if 
sufficient space be not allowed the full effect of this con- 



FlG. 2. 




Baker's New Student's Microscope. 



trivance is not experienced. It is most certainly desirable 
to have a condenser ; the search for micro-organisms has 
now become almost a matter of routine in certain cases 



14 MEDICAL MICROSCOPY. 

of doubtful diagnosis, and for their sure detection a special 
illumination is absolutely necessary. 

The diaphragm is made of several different patterns. 
The most ordinary form consists of a '^ wheel" of dia- 
phragms of various sizes, and should be examined to see 
whether the apertures are concentric^ with the axis of the 
instrument. Another form consists of small blocks in 
which the openings are pierced, all fitting into a common 
receptacle, which is inserted below the stage. The most 
important feature is that the diaphragm must be placed 
hfimediately beneath the object, in order to secure the best 
results. 

There are several more complicated forms, such as the 
*^ iris," but for a description of them the reader is referred 
to larger treatises on microscopy. 

Although the choice of a '^ stand" is of no mean im- 
portance, the chief point is of course the selection of the 
objectives. 

For ordinary histological work, an *'inch " lens and a 
^' quarter-inch " is sufficient, but if it be intended to under- 
take the examination of sputum, etc., a twelfth-oil-immer- 
sion is necessary, as the micro-organisms cannot be seen 
with any certainty without this power. 

According to the Continental systems, the first named 
lenses are represented by Nos. 3 and 7 of Hartnach, or A 
and D of Zeiss. 

It should be borne in mind that the term '' one-inch 
objective," etc., indicates only that such a lens should 
possess the same magnifying power as a single lens of one- 
inch focus, and that it does not refer to the distance which 
the front of the lens focuses from the object, this varying 
greatly with the different makers. 

The qualities of a good objective are: — defining 



THE MICROSCOPE AND ACCESSORIES. 



power, flatness of field, penetrating power, and resolving 
power. 

Defining power. — Expressed in more exact language 

Fig. 3. 




Beck's Pathological Microscope. 

this means that the objective has been properly corrected 
for spherical and chromatic aberration. 

** Spherical aberration" causes the lines of the 



1 6 MEDICAL MICROSCOPY. 

object to appear blurred and foggy, instead of being 
distinctly defined and clearly separated from one another. 
If the glass be not free from chromatic aberration, 
colored fringes appear around certain spots, blue, if the 
lens be under-corrected, red, if over-corrected. ** Defin- 
ing power" may be tested by examining a stage-micro- 
meter and observing if each division can be clearly 
distinguished. 

Flatness of field. — If an object be moved about from 
one portion of the field to another, it ought to appear 
equally well-defined in all parts. Thus, taking the stage- 
micrometer again as a test, the lines should be sharp and 
clear, and perfectly parallel to one another in every part 
of the field. 

Penetrating power. — By this term is signified the 
power of an objective object to view several planes of an 
object without altering the focus. A section of liver is a 
good test for judging this properly. 

The penetrating power of a lens is dependent on its 
** angle of aperture." This is the angle made by two 
lines drawn from opposite sides of the aperture of the 
object-glass with the principle focus of lens. 

If the system of lenses has a '^ low angle," its penetrating 
power is greater than one which possesses a '* high angle " 
of aperture. 

In connection with this subject it may be mentioned 
that low-power objectives with a high-angle and conse- 
quent shorter working distance will define much better 
than those with smaller apertures. 

Resolving power. — This again depends entirely upon 
the aperture, supposing the glass to be perfect in other 
respects; for good effects the angle ought to be large. 
Some authorities object to the term *^ resolving power" 
as being synonymous with *' defining power." An object- 



Fig. 4. 




Swift's Bacteriological Microscope. 



1 8 MEDICAL MICROSCOPY, 

ive, however, may have perfect defining power, and yet 
by reason of its want of aperture it will be unable to dis- 
tinguish certain minutiae. It defines all that it can take 
up, but cannot define what is not imaged by it. 

In choosing an objective, it is well to select one with an 
universal screw, so that it can be used with any stand, 
otherwise an ^^ adapter " will become necessary. 

A *' nose-piece *' (Fig. 5) carrying two or three ob- 
jectives is a useful adjunct, saving a great deal of time when 

Fig. 5. 




Nose-piece. 

working with more than one power. This little piece of 
apparatus should be tested to ensure of there being sufficient 
space for the oil-immersion to revolve between the tube and 
the stage, and some minute object should be selected by 
means of the low power and arranged so that it occupies 
the centre of the field ; the high power should tlien be 
turned round and lowered on to the glass, when this 
particular object should still be in the centre of the field ; 
if this be not the case, the nose-piece is not correctly 
** centred " and should be rejected. 



THE MICROSCOPE AND ACCESSORIES. 1 9 

Not more than two oculars, a high and low, will be 
required ; their function, as already stated, is to magnify 
the image formed by the objective ; but enlargement is 
better obtained by drawing out the tube, or by substituting 
a stronger lens, than by using a high eye-piece, which is 
trying to the eyes. 

A Camera-lucida is also of service for drawing the 
outline of specimens; one need not be bought, however, 
for it is easy to manufacture one by bending a small piece 
of tin-foil round the upper end of the tube, and arranging 
it in such a fashion as to support a cover-glass at a proper 
angle to the eye-piece ; the angle is readily ascertained by 
experiment, and this simple contrivance has the advantage 
over the more complicated ones, in that the glass being so 
thin, only one image of the pencil instead of two is seen, 
and it is consequently much easier to use. This is the 
principle of Beale's ^^ neutral tint reflector." 

It would be invidious and beyond the function of this 
book to recommend any particular maker from whom a 
microscope should be bought. There are many excellent 
dealers both in London and the Provinces. It is presumed 
that those who make use of these directions will not be 
desirous of possessing a large and expensive instrument. 
Very good '^students' microscopes" are now made by 
many of the London firms^ which can be highly recom- 
mended. They are fitted with all the apparatus necessary 
for simple bacteriological work, and the cost is from ^16 
to ;^r2o. 

Any of the following makers may be fully trusted to 
supply good instruments, and any omissions that occur 
do not imply that therefore other firms are not equally 
as reliable : — 

C. Baker, 243 and 244 High Holborn. 

Beck, 68 Cornhill. 



20 MEDICAL MICROSCOPY. 

Crouch, 66 Barbican. 

Pillischer, ^^ New Bond Street. 

Powell & Lealand, 170 Euston Road. 

Swift, 81 Tottenham Court Road. 

Parkes, 5 St. Mary's Row, Birmingham. 

Ross, 164 New Bond Street. 

In America very similar instruments, possessing every- 
thing that can be desired, and excellent both in manu- 
facture and in the qualities of the lenses, may be obtained 
from Joseph Zentmayer, 209 South Eleventh Street, Phila- 
delphia, and Bausch & Lamb Optical Company, 48 and 
50 Maiden Lane, New York, and at 543 North St. Paul 
Street, Rochester, N. Y. These firms also supply all 
microscopical apparatus and accessories. 

If a suitable stand be already to hand, the extra appa- 
ratus may, of course, be added. This applies more par- 
ticularly to an oil-immersion lens and Abbe's condenser. 
The best immersions are perhaps those of Zeiss, but they 
are very expensive. A very good lens is made by Leitz, 
and costs about ^^5, whilst a condenser can be fitted for 
about ;^3. Both the lens and condenser may be obtained 
from any of the makers above mentioned. 

Having detailed the form of the microscope and the 
essentials of a good and practical one, we now pass to 
the use of the instrument. This may seem somewhat 
superfluous, but a few hints to the inexperienced may save 
them not only various petty annoyances, but probably also 
the loss of valuable lenses. 

In removing the instrument from its box it should be 
grasped by the column, not by its foot, as thereby much 
strain on its bearings is prevented. Any jarring of the 
adjustments soon puts them out of order. For this reason 
when in constant use it is preferable to keep it under a bell- 
jar rather than to be constantly lifting it out of and into 



THE MICROSCOPE AND ACCESSORIES. 21 

its case. Scrupulous cleanliness cannot be too strongly 

Fig. 6. 




Bausch & Lamb's Universal Microscope. 



2 2 MEDICAL MICROSCOPY. 

insisted upon. Each time the lenses are used they should 
be carefully wiped with a soft piece of chamois leather. 
This applies more particularly to the oil-immersion lens, 
for if the oil be allowed to remain on any length of time 
it is apt to become hard, and great trouble is experienced 
in removing it. 

It not infrequently happens when working with the high 
powers that these are accidentally forced through the cover- 
glasses and so become covered with balsam ; this is best 
removed by moistening the chamois leather with a little 
alcohol or xylol, rapidly passing it over the lens and im- 
mediately drying it. This must be done very cautiously, 
as the lenses are ^^ set " in balsam, and there is some danger 
of this being dissolved by the spirit, and if such be the 
case, the damage is extremely difficult to rectify. 

Having settled the microscope on a firm table or stand 
(for any vibration is very disturbing) and inclined it at an 
angle to suit the convenience of the observer, without 
causing him to adopt an uncomfortable or tiring posture, 
the mirror is turned so as to reflect the light up the tube. 
The best illumination is obtained with a North light, so as 
to avoid the direct glare of the sun, which is trying to the 
eyes ; a very good light is obtained by reflection from a 
white cloud. If a low power is being used the flat side of 
the mirror should be employed, and if a high one, the con- 
cave side. 

In conjunction with the mirrors, various diaphragms 
are used to regulate the quantity of light passing to the 
specimen. In examining unstained sections, or objects 
such as are found in urinary deposits, the diaphragm with 
the smallest aperture that can illuminate the whole of the 
field is applied. With stained specimens no regulation of 
the amount of light is necessary. 

With regard to the selection of a power to view any 



THE MICROSCOPE AND ACCESSORIES. 23 

particular specimen, the rule is, never use a higher power 
than is absolutely necessary for clearly bringing out the 
minutiae of the object under examination. The inch lens 
should therefore first be used to gain a general idea of its 
structure and nature, and then the quarter-inch may be 
substituted ; for ordinary medical work a higher power will 
not often be needed, except for the examination of micro- 
organisms. In using the high powers, in order to avoid 
breaking the cover-glass by injudicious use of the coarse 
adjustment, it is a good plan before applying the eye to the 
tube to lower the lens until it just touches the cover-glass, 
and then to work it upward by means of the fine adjust- 
ment whilst looking down the microscope; if the structure 
be very fine, it is advisable to slightly move the slide up 
and down with the left hand, as a moving object is more 
easily caught sight of than one which is still. 

It should be remembered that the magnifying power may 
be augmented : — (i) by increasing the power of the object- 
glass ; (2) by using a higher eye-piece ; (3) by increasing 
the distance between the object-glass and the eye-piece, 
that is, by drawing out the tube. 

In using the oil-immersion a drop of thick cedar oil is 
placed on the cover-glass, and the lens lowered until it 
comes into contact with the oil. The fine adjustment is 
then cautiously turned until the object comes into focus. 
At the same time care must be taken that the oil does not 
flow over the cover-glass and dissolve the balsam in which 
the section is mounted ; to prevent this it is a good plan to 
ring the specimen with gold size. 

The Abbe's condenser (Fig. 7) must be focused so as to 
produce the brightest possible illumination. The principle 
of the condenser is this : — It is of such a form that the rays 
of light reflected from the mirror pass through it in such a 
direction that their convergence forms a cone of light as 



24 



MEDICAL MICROSCOPY. 



highly concentrated as possible. Under these circumstances 
the outlines of unstained preparations appear blurred, 
and this illumination is consequently unsuited for fresh 
specimens. Also, with stained preparations the structure 
of the tissue or whatever may be under examination cannot 
be distinctly made out, but the portions which are more 
deeply stained, such as the nuclei, are brought clearly into 

Fig. 7. 




Abbe Chromatic Condenser. 



view. It is for this reason that the condenser is so valuable 
in searching for micro-organisms ; after the use of suitable 
stains the small rods and micrococci stand prominently out 
from the indistinctly defined back-ground and thus can 
readily be seen and recognized. 

Ifdiaphragms are placed between the mirror and condenser 
the characteristic action is cut off, and a good illumination 
is obtained for histological work and also for the examina- 



THE MICROSCOPE AND ACCESSORIES. 25 

tion of unstained micro-organisms, as in *'drop cultures." 
The same effect is also produced by lowering the condenser 
by means of screws provided for the purpose. 

A caution may be given here on the use of the nose- 
piece. In causing this mechanism to revolve, care must 
be taken to raise the tube high enough to allow the high 
powers to travel without touching the glass, otherwise, of 
course, the object will be moved, and thus the great advan- 
tage of the nose-piece frustrated. This is particularly liable 
to occur after searching the specimen for any one spot with 
a lower power, which it is desired to investigate with the 
oil-immersion lens, as in examining tissues for tubercle 
bacilli, etc. 

The two clips may be removed for ordinary work, but 
when using the immersion lens it is a good plan to take 
out the left-hand one only, leaving the right to fix one 
end of the slide whilst the other is moved about with the 
fingers. 

The chief use of the clips is to fix the slide in one 
position, either for purposes of demonstration or when 
drawing with or without the camera-lucida. 

It is not always possible to work by daylight, and conse- 
quently some form of artificial light must be employed. 
Nothing equals an oil-lamp. Various kinds of *^ micro- 
scopic lamps" (Fig. 8) are sold, which have in themselves 
no special advantages except in conjunction with very 
high powers, when, by means of a slit in the shade, only 
a thin stream of light is allowed to fall on the mirror, so 
producing more intense but localized illumination. For 
purposes of laboratory work at night, as well as for use 
with the microscope, ^^The Queen's" reading lamp can 
be confidently recommended. The common colza or any 
mineral oil may be burnt ; it is excellent as regards bril- 
liancy and steadiness of light. 
3 



26 



MEDICAL MICROSCOPY. 



With the modern improvements in electric-lighting it is 
probable that this mode of illuminating dwelling-houses 
will soon become more general, and this pure white light 
is very good for microscopic work, although it is often 
rather difficult to avoid the reflection of the incandescent 
wires. 

A good deal of discussion has taken place as regards the 

Fig. 8. 




Microscopic Lamp. 



correct manipulation of the mirror, especially when work- 
ing with the condenser, as reflections of the window or 
of the lamp are extremely apt to appear in the field. The 
only way to remedy this is to turn the mirror so as just to 
cause these images to escape the circle of the lens, this 
giving the next best light to the centre of convergence of 
the rays. 



THE MICROSCOPE AND ACCESSORIES. 27 

The next point to be considered in connection with the 
use of the microscope is the representation on paper of 
what is seen through the tube. This is naturally an im- 
portant matter, as it is almost impossible to give such a 
graphic description of the microscopical specimen as shall 
enable every one who reads it to make a mental picture of 
what the writer means. Time is not wasted, therefore, 
which is spent in practicing drawing objects, for the pains 
taken wall be amply repaid. 

The most simple method is to place a sheet of paper on 
the right of the microscope, and then to look down the 
tube with the left eye, keeping the right open gazing 
at the paper ; an image of the object will then appear to 
be thrown on the paper, and the outlines may be traced 
with a pencil. If the paper be placed on a level with the 
stage, the picture when drawn will be of the same size as 
the image seen through the microscope. If it be placed 
higher, the representation will be smaller, whilst if lower, 
then the drawing will give an enlarged outline of the 
field. 

A more exact copy is obtained by aid of the camera- 
lucida. There are several different forms of this instru- 
ment j the cheapest and most simple has already been 
described on p. 19, and this contrivance will answer all the 
purposes of the more complex ones as sold by the different 
makers. In using it, the glass slide is fixed in position by 
means of the clips, and the object accurately focused. 
The tube is then inclined at right angles to the stand, and 
the mirror arranged so that the object is illuminated, but 
not too brightly. The paper, placed so that the projected 
image shall fall upon it, should not be too much in the 
shade. Only the principal outlines need be traced, the 
shading and minutiae being filled in freehand. The draw- 
ing is best made with a hard pencil. 



28 



MEDICAL MICROSCOPY. 



Two Other forms of the camera-lucida are shown in 
the accompanying figures. Fig. 9 is a simple prismatic 
arrangement, but quite effectual. Fig. 10 is the instru- 

FiG. 9. 




Prismatic Camera-lucida. 



Fig. 10. 




Abbe's Camera-lucida. 



ment introduced by Abbe, and possesses the advantage 
that it may be used with the microscope in an upright 
condition. 



THE MICROSCOPE AND ACCESSORIES. 



29 



Another operation, which is, however, but rarely required 
in ordinary medical work, is ascertaining the size of an 
object. An appliance is employed known as the '* microm- 
eter." The form in most common use is the '^stage- 
micrometer," which consists of a glass slip on which is 
ruled a series of lines y^ of an inch apart, one of these 
divisions being again divided into ten parts. 

The most simple method of measuring an object is by 
means of a cover-glass made to act as a camera-lucida as 
above described. A stage-micrometer is placed under the 
microscope and focused ; the instrument being horizontal, 
a piece of paper is laid on a level with the stage, and the 

Fig. II. 




Eye-piece Micrometer. 



lines magnified by a quarter-inch lens are carefully traced. 
The micrometer is now removed and replaced by the 
object whose size it is desired to ascertain. The object 
is traced, and compared with the scale by the aid of 
compasses. 

Some microscopes are provided with an '^ eye-piece 
micrometer" (Fig. 11), a flat piece of glass having a 
scale ruled upon it inserted in an ordinary ocular. The 
value of the divisions of the scale must first be determined 
for each objective by observation of a stage micrometer, 
and this being once done, a note should be made for future 
use. In subsequently using it for measurement all that is 



30 MEDICAL MICROSCOPY. 

necessary is to see how many divisions of the scale the 
specimen under examination covers. 

A table is usually provided with evtry microscope 
designating the magnifying power of each objective with 
the different eye-pieces. Should this have been mislaid, 
or a new lens been added, the magnifying power may be 
determined as follows : — 

The stage-micrometer is used, and its image cast by 
means of the camera-lucida on a sheet of paper, placed 
exactly ten inches from the eye. Two lines, which are in 
reality j-^-q-q of an inch from each other, are then traced, 
and the distance between them on the paper accurately 
measured ; then the magnifying power is ascertained by 
calculating how many times the y-oVo ^^ ^^ ^^^^ ^^^^ g^ 
into the distance on the paper. Thus, supposing with an 
inch lens the lines of the image are -^q of an inch apart, 
then, -^Q -f- YoVo" "^ ^^° ^ therefore the system (objective 
and eye-piece) magnifies two hundred times. 

This completes a description of the use of the microscope, 
and we now pass to consider the accessory apparatus 
which is required. 

It need not be supposed that a separate room is needful 
for microscopic work. A laboratory is, of course, to be 
preferred, but all that is really requisite is a firm table and 
a good light. The table is best when made of deal ; it 
should stand on four steady legs, and be of such a height 
that the operator may sit comfortably before it while at 
work. It is essential that the edges of the table should be 
square, for if they are beveled it will be found impossible 
to affix the microtome to the bench by means of a clamp. 

For convenience in manipulating specimens it will be 
found a good plan to paint on the table close by the worker 
two oblong patches, one black and the other white, each 
being about a foot long and six inches wide, the former 



THE MICROSCOPE AND ACCESSORIES. 3 1 

to be used when dealing with unstained sections and the 
latter with colored. 

Convenient drawers or shelves should be close at hand in 
which to place the specimens to preserve them from dust 
and guard them from possible injury. 

If in the room chosen for this work there be no tap and 
sink, a large jug for water and a receptacle for waste must 
be provided. Vessels made of metal should be avoided, as 
they rapidly become corroded by acid, and therefore 
earthenware ones should be selected. 

It is almost impossible to prepare sections without the 
use of some special instrument. After considerable practice, 
sections, indeed, may be cut with a razor alone, but they are 
not so satisfactory, and even the most skillful worker can 
only succeed in procuring small ones, whilst by the aid of a 
microtome large and regular specimens may be obtained. 
Of these microtomes there are several kinds, varying in price 
from a few shillings to several pounds. 

For ordinary use either Frazer's modification of Cathcart's 
microtome or the instrument introduced by Swift may be 
safely recommended. 

Cathcart's microtome (Fig. 12) consists of a brass 
plate supported on a micrometer screw. The plate is set 
between two parallel wooden uprights, on which are fixed 
thick glass slides. These supports are about six inches long 
and three inches high. By means of projections the 
apparatus may be firmly fastened to the table by clamps 
provided for the purpose. The cutting instrument is a 
large plane-like knife set in a wooden handle. It looks 
very clumsy, but answers every purpose admirably. 

When it is used for preparing frozen specimens a spray 
apparatus is fixed beneath the clamp, the free tube being 
connected with a bottle of ether which is hung on the side 
of the microtome. 



32 



MEDICAL MICROSCOPY. 



When required for ^^ embedded '' specimens the brass 
disk is removed, and in its place a clamp (Fig. 13) is sub- 
stituted for the reception of the cork, on which the substance 
to be cut is fixed. 

Fig. 12. 




Frazer's Modification ot Cathcart's Microtome. 



Swift* s microtome (Fig. 14) has a large metal clamp 
for affixing it to the table surmounted by a thick round 
plate of glass in the centre of which is a small brass disk 
roughened on its upper surface. The spray apparatus, which 
is also connected with a bottle for containing ether, is fixed 



THE MICROSCOPE AND ACCESSORIES. 



33 



below this, and Swift has lately devised an arrangement by 
which a clamp may be substituted when embedded sections 
are being dealt with. 

With the microtome is supplied a ^* plough" for holding 
the razor. This is a tripod metal stand supported by 
screws, one at each angle. The one at the apex acts as a 
micrometer screw, and when turned, raises or lowers the 
edge of the knife. At the sides of the triangle are two 
smaller screws with notches at the lower ends. In the base 
of the tripod is another small screw, to which is attached a 
grooved block. This is for grasping the back of the razor, 

Fig. 13. 




Clamp of Cathcart's Microtome. 



whilst its edge fits into the notches already mentioned. By 
tightening these three screws the razor is firmly fixed. 

A freezing microtome in common use, especially where 
a number of specimens are required for demonstration 
purposes, is known as Williams' freezing microtome 
(Fig. 15). It consists of a round wooden box, provided 
with a drain pipe, in which the freezing mixture is placed. 
In the centre of the box is a vertical brass pillar, provided 
at the top with a roughened brass plate. The box is 
covered with a lid formed of a thick plate of glass, in the 
centre of which is a hole through which projects the plate 
4 



34 MEDICAL MICROSCOPY. 

for the reception of the specimens. The cutting instru- 

FiG. 14. 




Williams' Ether Microtome (" Swift's Microtome ").. 

ment used in connection with this microtome is Swift's 
^' plough " just described. 



THE MICROSCOPE AND ACCESSORIES. 



35 



Other microtomes are those of Katsch, Roy, Schanze, 
Zeiss, Jung, etc. If it be intended to do much section- 
cutting, one cannot do better than at once purchase the 
more complete instrument made by Reichert. It is more 

Fig. 15. 




Williams' Ice and Salt Microtome. 



expensive, costing about ^^6, but is far better adapted for 
cutting '* embedded" specimens. 

The knife is fixed on a carriage which runs in a groove, 
and its movement is, therefore, very steady. The micro- 
meter screw is worked automatically. Its general form will 
be sufficiently appreciated by referring to the accompanying 



;^6 MEDICAL MICROSCOPY. 

illustration (Fig. i6). M is the razor fixed to the block K, 
by means of the clamp F. By knocking against the screw 
c, fixed by the milled head ^ to the carriage by means of 
a spring arrangement^ the wheel Z is turned round and so 
raises the clamp g in which the specimen to be cut is held. 
This clamp is fixed by the screw e. An indicator / marks 
the amount which the object is raised each time the lever 
h is set in action. ^ is a catch regulating the movement 
of the wheel Z, while Wis sl metal trap for catching the 
waste alcohol. 

A very capital instrument has been introduced by the 
Cambridge Scientific Instrument Co., known as the rock- 
ing microtome (Fig. 17). Its chief use is for cutting a 
series of sections, but for ordinary purposes it has no 
advantage over the instruments already described. 

Swift has recently introduced an ingenious modification 
of the rocking microtome, in which clamps are provided 
so that it may also be used for cutting specimens embedded 
in celloidin, and also as a freezing microtome. 

It is often useful to be able to cut sections of fresh 
material without any preliminary preparation, such as 
during an operation for the recognition of the different 
forms of tumor. For this purpose nothing is better than a 
Valentin's knife (Fig. 18). It is formed of two narrow 
blades lying parallel to each other, their distance being 
regulated by a fine screw. 

The method of using the above instruments will be 
described when directions are being given for cutting 
sections. 

A certain number of accessories for convenience in 
microscopic work will be wanted, but these, however, 
require little explanation beyond enumerating those ab- 
solutely needed. Objects are nearly alv/ays examined 



THE MICROSCOPE AND ACCESSORIES. 



37 




38 



MEDICAL MICROSCOPY. 



on glass slides about three inches long and one inch 
wide. The edges are usually ground, but in a cheaper 







make they are rough. It is scarcely necessary to say that 
the glass should be quite free from specks and flaws of all 



THE MICROSCOPE AND ACCESSORIES. 39 

kinds. The specimens are covered by thin squares or 
circles of glass known as cover-glasses. These are 
made in varying thicknesses; the most useful are the 
squares, ^ of an inch wide, and the thickness known as 
*^ No. 2." These glasses are sufficiently thin to be used 
with a ^-inch lens and the oil immersion ; if, however, 
an eighth dry power be used, thickness '* No. i " must be 
employed. Great care must be exercised in cleaning the 
cover glass, as, being extremely brittle, they are easily 
damaged. 

When first purchased they are often covered with a 
thick film of grease which is difficult to remove. The 
best mode of doing this is to place them for a short time 
in strong hydrochloric acid, then swill them in water, and 

Fig. 18. 

28 



Valentin's Knife. 

finally transfer them to a covered capsule containing 
methylated spirit ; and here they should be kept until 
required. Before use they are dried with a soft cloth, 
being rubbed between the finger and thumb, or a corner 
of the cloth is laid on the table, the cover-glass placed on 
it, and the glass is gently wiped on either side. ^^ No. i '* 
cover-glasses being very thin, a good plan is to swill them 
in absolute alcohol as a final stage in the cleaning; this 
evaporates quickly, and does away with the need of a 
cloth. 

Cover-glasses which have been used may be recleaned in 
the manner above described. A tumbler or beaker 
covered with a ground glass plate should be kept on the 
bench, into which all waste alcohol should be thrown. 



40 MEDICAL MICROSCOPY. 

Specimens which have been mounted in balsam, but which 
have not been considered worth preserving, can be placed 
in this tumbler, and cover-glasses dissolved off. 

Some fine sewing needles should be mounted in 
wooden handles, and must always be kept clean and sharp. 
Special holders are sold in which the needles may be con- 
veniently changed when they are blunted or spoiled. 

Excellent substitutes for needles are glass rods drawn 
out to a very fine point. These are superior to needles in 
that they do not corrode in acids, and are not so apt to 
tear the specimens. 

Two pairs of forceps will also be required ] those 
should be selected which have broad blades, so that cover- 
glasses can safely be grasped by them, and the student 
should avoid the long, slender forceps which are usually 
sold with cases of microscopic instruments, as they are apt 
to chip and break the glasses. 

A scalpel and pair of scissors are also necessary, as 
also a couple of section lifters. The last named are 
preferably made of some mareable material, such as stout 
tin foil. 

Of glass apparatus the following are required. About 
half a dozen capsules of different sizes, the same number 
of watch glasses with ground bottoms, to ensure their 
steadiness, and two glass funnels. 

For holding the stains when in use nothing answers 
better than small glass salt-cellars ; they do not easily 
upset, and are of a convenient depth. 

A packet of filter paper and a filter stand w^ill com-, 
plete the apparatus for ordinary microscopic work, whilst 
for the examination of urinary deposits, etc., test-tubes, 
some conical glasses, and pipettes will be required. 

For the reception of the reagents, bottles with glass 
stoppers are preferable. 



THE MICROSCOPE AND ACCESSORIES. 4I 

A large supply of stains and other reagents is not neces- 
sary, and the following is a list of those absolutely needful. 
Those required for special purposes will be described as 
the need for them occurs, or they can always be added 
from time to time. 

Absolute alcohol. 

Methylated spirit. 

Glycerine. 

Farrant's solution. 

Canada balsam dissolved in xylol. 

Normal saline solution. 

This is made in the following manner (Woodhead) : — 
Sodium chloride is heated to redness, and cooled over sul- 
phuric acid ; 7}^ parts by weight are then dissolved in 
1000 parts by measure of distilled water. 

Iodine dissolved in iodide of potassium. — The 
best formula for this is that known as ^* Gram's solution." 
This not only acts as a useful testing fluid, but is also an 
essential part of the process for staining certain micro- 
organisms. 

It is made as follows : — 

Iodine, I part, 

Iodide of potassium, ..... 2 parts, 
Distilled water, 300 parts. 



CHAPTER II. 
HARDENING AND DECALCIFYING. 

For examination by the microscope sections may be pre- 
pared in two ways, either directly from fresh specimens, 
or from those which have been duly ^Miardened." The 
former method is practically confined to the investigation 
of tumors in the operating theatre, or for purposes of 
immediate diagnosis in the post-mortem room. When 
prepared in this manner, however, the sections cannot be 
permanently mounted, and hence are only of temporary 
service. A knowledge of how to prepare such sections is 
nevertheless greatly to be desired, and a chapter will be 
devoted to a consideration of this subject. • The processes 
by which specimens may be mounted, and kept for future 
reference, or for purposes of demonstration, will be first 
described. 

In selecting the portions of an organ or tissue for micro- 
scopical purposes, some care has to be exercised. They 
should be obtained as soon after death as possible, for if 
decomposition has set in the sections will not properly 
take up the stain, and thus many important characteristics 
may be lost. In choosing the parts of a diseased organ, 
the liver, for example, it is always preferable to take a par- 
ticle from near the surface, so as to include the capsule, as 
this affords great aid in recognizing the minute anatomy of 
the organ. Again, in preparing sections of the intestine 
or stomach, small portions should be cut off transversely 
to the axis of the viscus and about a quarter of an inch 

42 



HARDENING AND DECALCIFYING. 43 

in length, so as to show the condition of its various 
coats. 

When investigating a morbid growth invading an organ 
at least two pieces should be cut out — one to be taken from 
the centre of the tumor, and one from its circumference, 
so as to include a portion of the organ as well as of the 
growth. 

The size of the pieces should be nearly an inch in 
length and breadth, and a little less than a quarter of an 
inch in thickness. They should be removed by a sharp 
scalpel, the cuts being cleanly made so as to avoid any 
tearing, and when completely freed should be lifted upon 
the knife and' dropped into a small, wide-mouthed bottle, 
which should be at once labeled with the name of the 
organ from which the specimen was taken, the date, and 
supposed morbid condition. Too much stress cannot be 
laid upon the importance of immediately labeling all 
preparations, whether completed or otherwise, as from 
postponing this simple precaution many errors are apt to 
arise, and cause waste of time and much annoyance. 
Added to which a good specimen is of little value unless 
its source, in addition to its nature, is known. 

The bottle into which the cuttings have been placed 
should be filled with the hardening solution, and the choice 
of such a solution must now be considered. 

The reagents in most common use are : — 

Absolute alcohol, 
Methylated spirit, 
Miiller's fluid, and bichromate of potash. 

The selection of one of these depends upon many points. 

(a) The Organ or Tissue which is to be Hardened, — The 
most useful reagent is undoubtedly absolute alcohol. It 
is suitable for nearly all tissues, except those which, owing 



44 MEDICAL MICROSCOPY. 

to their complex structure, do not contract regularly. 
Thus, the central nervous system and the eye should not 
be subjected to its action. Thin tissues and membranes 
also are best hardened by some other fluid, on account of 
the manner in which in the alcohol they curl up ; for 
instance, sections of the intestines cannot be satisfactorily 
obtained after hardening in this reagent. For all such 
exceptions Mliller's fluid is preferable. 

(/^) The Nature of the Morbid Change. — Alcohol, owing 
to its solvent power on fat, should not be used when it is 
desired to demonstrate fatty changes ; on the other hand, 
it is most suitable when micro-chemical reactions are to 
be exhibited, such as the amyloid reaction. When speci- 
mens are hardened in Mliller's fluid or bichromate of 
potash, such reactions are marred. 

{/) It is occasionally desired to investigate certain par- 
ticular elements of the tissue, such as the epithelial struc- 
ture-, or the division of nuclei. For these purposes it is 
advisable to use the chrome salts. 

{^d^ For bacteriological purposes absolute alcohol or 
methylated spirit should be used, as the other reagents, 
although they do not entirely destroy, yet greatly interfere 
with the properties which the various bacteria possess of 
retaining the aniline dyes. 

{e) The time which the different hardening reagents 
occupy before the specimens are ready for cutting is occa- 
sionally of importance. If it be desired to secure a rapid 
and energetic action, provided there are no other objec- 
tions, absolute alcohol should be chosen ; whereas, if a 
more cautious course is advisable, Mliller's fluid or bichro- 
mate of potash should be selected. 

Before describing seriatim the method of using the 
above named hardening reagents, a few general hints 
may be given. A relatively large volume of the liquid 



HARDENING AND DECALCIFYING. 45 

should be employed and frequently changed ; the periods 
for so doing depending upon the fluid used. As before 
stated, the specimens should be placed in the reagent as 
soon as possible after removal from the body. They 
may either be suspended by means of threads in a tall 
cylindrical vessel, each thread having attached to it a 
label, by means of which it may be easily recognized, 
or a layer of cotton-wool may be placed in the jar ; 
the reason for these precautions being, that the specimens 
may be entirely surrounded by the hardening reagent. 

If Miiller's fluid or bichromate of potash are the re- 
agent selected, the jars or bottles should be kept in a cool, 
dark place, as these fluids are very apt to develop moulds. 

Absolute Alcohol. — This acts as a hardening re- 
agent by means of the withdrawal of water and coagula- 
tion of albumin. Specimens placed in it usually shrink 
somewhat, but this is of very little moment, for when 
the sections obtained from them are placed in water, 
they soon resume their natural appearance. 

As previously mentioned, it is suitable for nearly all 
structures, the exceptions being those which, owing to 
their varied composition, shrink unequally, such as the 
spinal cord and eye, but even these with a little manipu- 
lation may be hardened with this reagent. Thus, if it 
be desired to harden an eye-ball, this can be effected by 
injecting alcohol into the vitreous by means of a hypo- 
dermic needle before the specimen is placed in the jar ; 
no inequalities will then be produced. 

Again, lungs, muscles, and old spirit preparations are 
sometimes difficult to harden. This may be accom- 
plished by placing the specimens for twenty-four hours 
in equal parts of glycerine and mucilage of gum arabic, 
and transferring them once more to the alcohol, when 
an excellent consistence for cutting will be obtained. 



46 MEDICAL MICROSCOPY. 

The portions to be hardened should always be small, 
as, owing to the rapidity with which alcohol acts, the 
outer surfaces of the specimens become so set as to 
prevent the alcohol penetrating to the interior. 

The liquid generally becomes turbid a few hours after 
the fresh tissues are immersed in it, and therefore after 
twenty four hours it is better to transfer them to a fresh 
lot of alcohol, but subsequently no further change need 
be made, as there will be no tendency to over-harden. 
In this respect alcohol gives far less trouble than Mliller's 
fluid or bichromate of potash, and the hardening process 
is, moreover, more easily controlled, for the action of the 
last named reagents greatly depends upon time and 
temperature, and very often produces cloudiness and 
discolorations. 

As regards the time required for the completion of 
process, this greatly depends upon the size of the speci- 
mens. Small ones will be ready in twenty-four hours, 
whilst larger ones require about four days. 

Methylated Spirit. — This is suitable for the same 
class of objects as absolute alcohol, and its effects are 
practically the same. Its action, however, is not so 
rapid, and the specimens should remain immersed for 
about fourteen days. The spirit should be changed 
after the first twenty-four hours, and again after the 
first week. 

If time be no object, it is very suitable for hardening 
tissues in which it is intended to search for micro- 
organisms. 

Mliller's Fluid. — This has the following composi- 
tion : — 

Bichromate of potash, 2 parts, 

Sulphate of sodium, i part, 

Distilled water, loo parts. 



HARDENING AND DECALCIFYING. 47 

This reagent fixes the protoplasm of the cells rather than 
hardens them, and so, unlike alcohol, causes but little 
shrinking. 

It may be used for any tissue except those in which 
micro-organisms are to be sought for, or in which some 
micro-chemical reactions may have to be made use of — 
such as in staining amyloid material. There is one other 
rare exception which may be mentioned, a deposit of lime 
salts is dissolved by this medium, and consequently chalky 
deposits, such as occasionally occur in the glomeruli of 
the kidneys, are destroyed if the organs be hardened in 
this way. 

Mliller's fluid is especially adapted for hardening the cen- 
tral nervous system, and, indeed, is essential when Weigert's 
or Pal's methods of staining are adopted. Portions of 
intestine or other hollow organs are also best hardened 
by this fluid. Specimens of any size, even whole brains, 
may be prepared in this manner, but as the penetrating 
power of the liquid is very small, a considerable time must 
elapse before the process is complete. Even the smallest 
portions of any organ require about six weeks, whilst a 
brain must remain about a year. Moulds are likely to 
form, and to prevent this a small piece of camphor should 
be kept floating in it. 

The process of hardening may be hastened by placing 
the jars in an oven kept at a temperature of 30 to 40° C. 
(80 to 100° F.). The fluid has to be frequently changed, 
every second day for the first week, and then each week 
until the hardening is complete. In order to ascertain 
when this stage has been reached, the specimens should be 
removed from the jars and gently pressed with the fingers, 
when, if the desired result has been obtained, they will be 
found to be tough but not brittle. They are next placed 
in water, which should be frequently renewed until the 



48 MEDICAL MICROSCOPY. 

washings are colorless, and are finally put into methylated 
spirit until an opportunity is found for cutting them. 

Bichromate of Potash. — This salt is usually employed 
in the strength of from two to five per cent, watery solution, 
commencing with the former and gradually increasing the 
amount of the potash salt until the latter strength is ob- 
tained. It is used for the same class of specimens as 
Mliller's fluid, but its action is rather quicker, three 
weeks being required for specimens of ordinary size, but 
six weeks for a spinal cord, and six months for an entire 
brain. 

The after-treatment is the same with both reagents. 

Another form in which bichromate of potash is used is 
Erlicki's solution. 

The composition of this is as follows : — 

Bichromate of potash, 2.5 parts, 

Sulphate of copper, i part, 

Water, 100 parts. 

This reagent, owing to the presence of the sulphate of 
copper, hardens very much more rapidly than the previously 
described chrome preparations. A spinal cord may be 
hardened in it in ten days at ordinary temperatures, or in 
four days at a temperature of about 100° F. 

To give a complete list of chromic acid salts which are 
used for hardening purposes, the following must be men- 
tioned : — 

Chromic Acid. — This hardens extremely rapidly, 
most specimens being ready in a few days. The strength 
is gradually increased ; at first it should not exceed ^ per 
cent., and this should be gradually raised to ^ per cent. 
It is very important that small pieces of tissue should 
be placed in it, and large quantities of the solution 
employed. The fluid must be changed at the end of the 



HARDENING AND DECALCIFYING. 49 

first, second, and third days, and then every third day 
until the hardening is complete, this being tested at 
frequent intervals as already described, for if allowed to 
remain too long the specimens become brittle and almost 
useless. This may in part be prevented by adding a little 
glycerine to the reagent. Care must be taken to thoroughly 
wash out the acid with water before placing them in alcohol 
for preservation. 

Chromic Acid and Spirits. — This mixture, first 
introduced by Urban Pritchard, is occasionally employed 
instead of chromic acid. It is made thus : — 

Chromic acid, i part, 

Water, 20 parts. 

Rectified spirits, I So parts. 

The chromic acid must be dissolved in the water, and 
then the spirit added, otherwise a too violent action will 
ensue. 

This mixture is especially suited for hardening speci- 
mens of the eye or internal ear. 

Ammonium Chromate and Bichromate of 
Ammonia. — Both these salts are occasionally used as 
hardening reagents, and in a similar manner. They are 
suitable for all tissues, but the former is especially useful 
for preparing the kidney and other secretory glands. 
The strength of each is the same, /. ^., five per cent. 
The portions of tissues should be very small, and placed 
in about fifteen times their bulk of the solution. With 
ammonium chromate the process is complete in forty-eight 
hours, and the specimens are then thoroughly washed in 
water, and either cut at once or removed to spirit for 
preservation. With bichromate of ammonia the time 
occupied is much longer. The fluid must be changed at 
the end of the first, third, and seventh days, and at the 
* 5 



50 MEDICAL MICROSCOPY. 

end of each week until the sixth, when the tissues will 
be ready for cutting. 

It will be seen from the above descriptions that the 
chrome salts usually require a long time before the hard- 
ening process is complete, and as they penetrate slowly, 
this depends in a great measure upon the size of the speci- 
mens. Frequent changes are necessary, and therefore 
much more attention is required than when alcohol is 
used. Various alterations are also produced by their 
action in the interstices of the tissues. A peculiar net- 
work due to coagulation of albuminous substances very 
often takes place which may lead to errors if not rightly 
understood. Another disadvantage is that if the pieces 
are allowed to remain too long in the fluid they will be 
over-hardened and become brittle. 

Corrosive Sublimate. — This, though seldom used, 
is a capital hardening reagent. It is especially useful for 
glandular structures. The best strength is a half-saturated 
alcoholic solution ; this is made by dissolving in one por- 
tion as much of the salt as 70 per cent, alcohol will take 
up, and then adding to it an equal quantity of alcohol of 
the same strength. The specimens must not remain in it too 
long, as it hardens with great rapidity. A quarter of an 
hour to two hours (depending on the size of the speci- 
mens) is quite sufficient. At the end of the process the 
corrosive sublimate must be washed out with alcohol, 
otherwise the sections will be marred by the presence of 
black spots. 

Platinum Chloride. — This reagent is particularly suit- 
able for delicate objects such as the retina. It is usually 
made up in what is known as MerbreFs solution. This 
consists of equal volumes of 1.4 solution of chromic acid 
and 1.4 solution of platinum chloride. Its action is fairly 
rapid, the process being complete in from three to four 



HARDENING AND DECALCIFYING. 5 I 

days. No change is necessary, and the reagent should be 
washed out with dilute alcohol (60 per cent.). 

Osmic Acid. — Owing to its expense and the irritating 
property of its vapor, this substance is rarely used for 
hardening purposes, except in the Pal-Exner method of stain- 
ing the central nervous system and for portions of retina. 
It is sold as a I per cent, solution, and must be kept in 
opaque bottles in the dark. Specimens should be submitted 
to its action for about ten hours, and then thoroughly 
washed in water. One of the most important uses of osmic 
acid is the selective power which it has on fat cells, these 
becoming blackened. A good plan in demonstrating fatty 
changes is first to harden the specimens in MuUer's fluid 
and then to place them for a few hours in osmic acid. 
Another point which may be mentioned here is that 
turpentine and some other essential oils will decolorize 
particles of fat blackened by this reagent, and that, there- 
fore, when sections have been cut and stained, the pro- 
cess of ^* clearing" by these oils must be rapidly con- 
ducted. 

Picric Acid. — A saturated solution may be used. 
The albuminates are gradually transformed into the 
insoluble form, so that hardening occurs without any 
shrinking. At the same time the specimens are stained a 
deep yellow, which is very advantageous as a counter-stain 
to carmine. It is especially useful for epithelial structures, 
intestine, and the various tumors. After a stay of forty- 
eight hours in the acid, the specimens are washed in water 
and preserved in alcohol. Picric acid is occasionally em- 
ployed as Kleinenberg's solution. This is prepared as 
follows : — 

Saturated watery solution of picric acid, . . .100 parts, 

Strong sulphuric acid, . . 2 '^ 

Filter and add distilled water, 300 •' 



52 MEDICAL MICROSCOPY. 

This hardens more quickly than picric acid alone 
(three to twelve hours) and is especially adapted for the 
rapid hardening of various soft structures, such as sarcoma 
or myxoma. These sections should afterward be stained 
in carmine. 

DECALCIFYING. 

In order to prepare sections of bone or of tissues in 
which lime salts are deposited it is necessary to remove 
these in order that the substances may be cut with a razor, 
for the process of grinding down such specimens uatil 
they are thin enough to be properly mounted is far too 
tedious, and the results which are produced are unsatis- 
factory. 

The process of decalcification is carried out in much the 
same way as the process of hardening, except that, of 
course, the fluids employed are not quite the same. Only 
the principal reagents will be described here. 

Nitric Acid. — This is used in two strengths. For large 
bones one part of pure nitric acid is diluted with ten parts 
of water, whilst for smaller bones and for specimens con- 
taining calcareous deposits this must be further diluted to 
one per cent. 

Specimens are first treated with absolute alcohol for 
three days, and are then placed in the dilute nitric acid 
for eight or ten days, the fluid being changed daily. 
They must be frequently examined to ascertain if the 
process of decalcification is complete, this being tested by 
piercing them with a needle. When ready they are washed 
for some hours in water and then placed in alcohol for 
preservation. A modification of this process, and a very 
excellent one, consists in making a mixture of equal parts of 
three per cent, of nitric acid, and seventy per cent, of 
alcohol. This acts more slowly than the previous one, 
requiring several days or even weeks. 



HARDENING AND DECALCIFYING. 53 

Perenzi's Solution. — This is, perhaps, about the best 
decalcifying fluid. Its composition is as follows : — 

One-half per cent, chromic acid, 3 parts, 

Absolute alcohol, 3 " 

Ten per cent, nitric acid, 4 *' 

Its action is very rapid, the specimens being ready in 
one week. The after-treatment is the same as for nitric 
acid alone. The advantage of the three fluids just men- 
tioned over other decalcifying fluids is that no swelling is 
caused, whilst at the same time no injury is suffered by the 
tissue elements. 

Hydrochloric Acid. — The strength of this acid for 
decalcifying purposes is ten per cent. The process is much ' 
slower than with nitric acid, requiring a month before 
it is completed; in addition, the acid causes the soft 
tissues to swell, so that their structure becomes almost 
unrecognizable. To avoid this, a little chromic acid or 
alcohol may be added. 

Von Ebner's Solution. — This is a modification of 
the preceding, and has the following composition : — 

Hydrochloric acid, 5 parts, 

Alcohol, 1000 " 

Distilled water, 100 *' 

Chloride of sodium, 5 *' 

A large quantity of the fluid must always be used and 
frequently changed, the specimens being also occasionally 
tested to ascertain if they are soft enough for cutting; 
they are afterward thoroughly washed in water. If it be 
desired to examine the fibrillar structure of bone, the 
sections should be mounted in ten per cent, salt solution, 
but if this is not a matter of importance, the ordinary 
mounting media may be employed. 

Picric Acid. — A saturated watery solution of picric 



54 MEDICAL MICROSCOPY. 

acid may be used, but is not altogether satisfactory. 
Its action is weak and very slow; in addition, it causes 
considerable shrinking of the tissues. A large volume of 
the reagent must be employed, and a (qw crystals of the 
acid should always be kept at the bottom of the jar to 
insure the strength being maintained. When decalcifi- 
cation is accomplished, after washing in water, the speci- 
mens are preserved in spirit. This process is only suitable 
for very small pieces of young bone. 



CHAPTER III. 
EMBEDDING. 

In order that thin and uniform sections may be made it 
is not sufficient that the specimens have been hardened or 
decalcified. If it be attempted to cut the specimens with- 
out further preparation, it will be found that the sections 
easily break directly they are made, if, indeed, it be possible 
to obtain any at all. In addition to these objections it is 
very difficult to hold the specimens firmly enough without 
crushing them and greatly interfering with their structure. 

In order to overcome these obstacles, it is necessary 
either to surround the material with some substance firm 
enough to afford a hold to the hand, or to saturate it with 
a medium which will impart to it a uniform consistency. 
This operation is known as '^ embedding." 

From the above prefatory remarks it will be seen that 
embedding is necessarily of two kinds, viz., '* simple" 
embedding, which is fixing or placing the object to be cut 
in a medium firm enough to hold it, and '' interstitial " or 
*' infiltration " methods, which consist in permeating the 
specimens with some medium, which is at first fluid, but 
gradually becomes solid; this being accomplished by the 
use of substances which are liquid when hot and solid when 
cool, such as paraffin, or by dissolving some substance in a 
volatile fluid, such as celloidin in ether and alcohol. A 
third method, and the one which is perhaps the most com- 
monly adopted, consists in saturating the specimens with 
a solution of gum, which is afterward frozen. 

55 



56 MEDICAL MICROSCOPY. 

The freezing media will be first described. The 
initial stage consists in abstracting the hardening reagent. 
This is, of course, absolutely essential, as alcohol will not 
freeze, and the chrome salts would be deposited irregularly. 

The specimens, therefore, are placed in a basin contain- 
ing ordinary tap water, which should be frequently renewed. 
The process is shown to be complete by the specimens 
sinking to the bottom of the containing vessel if they have 
been hardened in alcohol ; or by the water remaining un- 
tinged if chromic acid or its salts have been used. 

They are now ready to be placed in gum. The solution 
usually employed is gum and syrup, which is made of 
equal parts of gum solution (B. P. strength) and syrup, to 
which a little thymol should be added as a preservative. 

The following, however, recommended by Mr. T. L. 
Webb (^The Microscopic Journal^ ix, 1889, page 344), 
will be found preferable. It freezes sufficiently to give 
a firm support without being too hard, it keeps better than 
gum, and is in addition cheaper. 

An aqueous solution of carbolic acid (i in 40) is taken 
and sufficient dextrin is dissolved in it to make a thick 
syrup. 

Dextrin dissolves slowly in cold water, so that a gentle 
heat must be used when making the mucilage. It solidi- 
fies if kept for some time, but may be liquefied again by 
gently warming the bottle. 

The specimens should be allowed to remain in these solu- 
tions for twenty-four hours, so as to become thoroughly 
permeated. 

SIMPLE EMBEDDING. 

Paraffin is usually employed for this method. 

A little tray is first made out of light cardboard in the 
following manner (Fig. 19). The card should be oblong 
in shape, as in the diagram. It is first folded along the 



EMBEDDING. 



57 



lines AA' and BB\ next along the lines CC and DD\ 
keeping the folds of the paper on the same side. The 
lines aa' , bb\ cc\ and dd' are then marked with a pencil. 
The corners are now pinched up between the finger and 
thumb and bent round so as to lie against the sides of the 
tray. The sides are secured by doubling tightly down 
the projecting corners. Two paraffins are usually em- 
ployed, a hard one melting at 49^ C, and a soft one 



Fig. 19. 





a' C 


B c^ 


A 


\ 1 

\ 

\ 


1 / 

j/ 


w 


c\ 


■R 


is 


>?! 




/i 

/ 

/ 


— - K - — — — — — 

i \ 

1 \ 
1 \ 






B' 



Diagram for Embedding Box. 



melting at 46° C. Two parts of the former are mixed 
with one of the latter, the resulting mass yielding a good 
consistency for cutting. If it be found too hard, a little 
vaseline may be added to soften it. 

A small piece of the paraffin mixture is heated over a 
water bath until the paraffin melts, and it is then poured 
into a cardboard tray. The specimen meanwhile must 
have been placed in alcohol and left there for a few hours. 
It is then removed and dried as far as possible by means of 
6 



58 MEDICAL MICROSCOPY. 

filter paper. As soon as the melted paraffin in the tray 
begins to set at the edges, the specimen is immersed in it 
and the mass allowed to solidify around it. When cool, 
a block of paraffin is cut out of the box, so as to contain 
the specimen and afford a convenient support whilst 
sections are being cut. 

INFILTRATION METHODS. 

In medical work, ^^ simple embedding" is very seldom 
employed ; it gives almost as much trouble as the methods 
now to be described, and the results are not nearly so good. 

The two chief media employed for the infiltration 
methods are celloidin and paraffin. In England the 
latter is more usually adopted, whilst abroad, and es- 
pecially in Vienna, celloidin is generally chosen. It is 
difficult to state which is the better of the two, but the 
celloidin method is more easy to learn, and the results are 
all that can be desired. It affords an excellent consistence 
for cutting, and at the same time is so transparent and 
flexible that it may be mounted with the specimen without 
detracting from the microscopical appearances; its only 
drawback is, that it is apt to retain some of the stains, 
especially the aniline dyes, but even this may be -obviated 
with a little care. If sufficient trouble be taken in the 
preparation of the solution, sections may be cut as thin as 
with paraffin, although it is generally stated that, when 
very thin sections are required, paraffin must be used. If 
both these methods are employed, a very good rule is cel- 
loidin for large, paraffin for small specimens. 

Embedding in Celloidin. — Celloidin is a preparation 
of pure pyroxylin. It can be obtained in two forms, in 
tablets or in cuttings, the latter being far more con- 
venient. The substance manufactured by Schering is the 
best. 



EMBEDDING. 59 

To prepare it for use, a few '^cuttings '* are placed in a 
small wide- necked bottle ; a mixture of absolute alcohol 
and ether, equal parts, is then poured in and allowed to 
remain for some hours, as the celloidin dissolves very 
slowly ; it should be stirred w^ith a glass rod from time to 
time, and sufficient of the mixed fluids added to form 
a mixture of about the consistence of thin mucilage. 

When dealing with very delicate specimens, such as 
spinal cord, the eye, embryos, etc., it will be found a good 
plan to prepare two solutions, one very fluid, and the 
other rather thicker, the specimens being placed first in 
the thin and then in the thick. 

If the specimens have been hardened in Miiller^s fluid - 
or any of the chrome preparations, this must be removed 
by thorough washing in water before they are removed to 
dilute spirit and then to absolute alcohol. If, on the other 
hand, they have been hardened in alcohol, the process of 
embedding may be at once proceeded with. The first 
stage consists in soaking the specimens in a mixture of 
equal parts of absolute alcohol and ether ; in this they 
must remain for about twelve hours. They are then trans- 
ferred to the celloidin mixture, if necessary, first to the 
thin and then to the thicker solution. Here again at 
least twelve hours' stay is necessary. Small blocks of cork 
are then prepared ; an ordinary bung cut into six pieces 
answers very well. It is important that one surface of the 
cork should be quite flat and perfectly dry. A large glass 
jar with a glass stopper will also be required ; this must be 
about half filled with eighty per cent, alcohol. 

After the specimens have been a sufficient length of time 
in the celloidin, they are removed one by one by means of 
forceps and each placed on a cork; a few minutes are 
allowed to elapse for the celloidin to begin to set, and the 
corks are then thrown into the jar containing the alcohol. 



6o MEDICAL MICROSCOPY. 

With a little manoeuvring, they can be arranged without 
contrivances to sink the corks, the weight of the speci- 
mens being sufficient for this purpose. 

They are allowed to remain in the alcohol until the 
sections can be cut, but a minimum time of twelve hours 
is necessary. 

Embedding in Paraffin. — As in the case of celloidin, 
the first stage of embedding is the saturation of objects 
with some solvent of the embedding material. There are 
various liquids recommended, turpentine, clove-oil, ben- 
zine, chloroform, xylol or cedar-oil. The latter has been 
highly praised as penetrating rapidly, mixing readily with 
the paraffin, and not tending to render the tissues brittle. 
Turpentine, however, is most commonly used, and except 
for very delicate structures, when cedar-oil is to be recom- 
mended, answers every purpose. 

The specimens are first dehydrated by means of absolute 
alcohol, in which they should remain for several hours. 

The specimens are placed directly from the alcohol 
into the turpentine or cedar-oil, and will be completely 
cleared in from one to six hours, according to their size. 

A mixture is then made of two parts of hard paraffin 
(melting at 49° C.) and one part soft (melting at 46° C), 
the mixture being placed in a small copper vessel or 
porcelain dish, and the paraffin melted and retained at a 
temperature of about 1° above its melting point; it is a 
matter of great importance not to heat the paraffin too 
much, but to keep it as nearly as possible at its melting 
point. It is this difficulty that renders celloidin preferable 
to paraffin. 

The objects are removed from the clearing mixture 
(turpentine, etc.) and immersed in the melted paraffin, 
where they should remain from three to five hours, when 
they will be found to be completely saturated. 



EMBEDDING. 6l 

The final stage in the paraffin process is the * 'embedding" 
proper. Small trays made in the way described above are 
filled with melted paraffin, and the objects are removed 
from the warm paraffin in which they have been lying and 
immersed in the liquid mass, and the whole should be 
cooled as quickly as possible by placing the trays in run- 
ning water, but not permitting them to be completely sub- 
merged. If the paraffin be allowed to cool slowly, there is 
great danger of its crystallizing, and so not forming a 
homogeneous mass. 

Instead of the trays, embedding L's may be used. 
These consist of two L-shaped pieces of lead, about half 
an inch high, the long arm being about two inches long' 
and the short one three-quarters of an inch. A glass slide 
is coated with glycerine, and the two pieces of lead laid on 
it so as to form a rectangular box, which is then filled with 
the melted paraffin. 

An extremely simple process, which may be adopted 
when the specimens are small, is to pour the melted mixed 
paraffins into a watch-glass or small porcelain cup, and 
then to place the specimens, after soaking in turpentine or 
chloroform, in the melted wax. The watch-glass or cup is 
then placed in an oven maintained at a temperature of 
about 52° (so that the paraffin is kept at about its melting 
point) for five or six hours. They are then removed and 
allowed to cool. When the embedding material has set, 
the glass is slightly warmed so as to loosen the wax, or the 
specimen may be cut out with a knife and the block pared, 
as described in the next chapter, ready for the microtome. 



CHAPTER IV. 
CUTTING SECTIONS. 

In the previous chapter the methods of hardening and 
embedding specimens have been described, and the next 
process is cutting the sections. 

For reasons already stated, at the present time sections 
are very rarely cut free hand, but generally by means of 
a microtome. The principal instruments have been 
described in Chapter I. It now remains to describe their 
use and their application to frozen and embedded objects. 

Except for special purposes, the method by freezing is 
the one generally adopted in this country ; it has the 
advantage of being very speedy, and by practice almost as 
good sections may be obtained by this means as by the 
more complicated processes. 

It is hardly applicable, however, to delicate specimens, 
such as the eye, and for bacteriological purposes it cannot be 
recommended, for the reason that when the gum has been 
frozen and melts again small particles are very apt to 
fall out. Take, for example, sections of tubercular lung. 
Excellent sections can be obtained by freezing the more 
solid parts where the fibrous tissue is in excess, but when 
breaking down of tissue has been going on, or when small 
caseous masses are present, it is extremely difficult to get 
good sections, owing to the fragile nature of the tissues, and 
the caseous nodules will probably be lost ; consequently, 
the search for tubercle bacilli is greatly interfered with^ and 
this drawback is added to by the multiplicity of fluids 

62 



CUTTING SECTIONS. 63 

through which the sections will have to pass before the 
staining is complete; for such specimens, therefore, it is 
advisable to embed in celloidin. In many of the Con- 
tinental laboratories freezing is but rarely resorted to, 
nearly all the specimens being embedded in celloidin, and 
the sections obtained are most valuable, far excelling in 
most respects the ordinary sections obtained by freezing, 
although, as before stated, by dint of long practice very 
good sections can be thus prepared. 

For certain purposes it is often desirable to cut sections 
in ^^ series," that is to say, to obtain a number of sections 
in the order in which they are cut, without losing any of 
the intermediate ones. It is possible to do this with the - 
ordinary microtomes, but not to nearly such perfection as 
can be done by the Cambridge Rocking Microtome. 
'^ Series " cutting, however, will be separately considered. 

We now proceed to describe in detail the methods 
of cutting sections, and we will first deal with frozen 
specimens. 

It will be presumed that all the foregoing processes for 
hardening, etc., have been duly carried out, and the 
objects which are to be cut placed in the gum. 

Cutting with Cathcart's Microtome. — One of 
the specimens is removed from the gum by means of 
forceps and placed on the plate of the microtome, a 
little of the mucilage being added so as to completely 
envelope it. The spray points having been set in position, 
the ether bottle is filled with ordinary methylated ether. 
The spray apparatus is then set to work, rather briskly at 
first, until a white ring forms at the edge of the gum, 
which gradually spreads until the whole is a frozen mass 
firmly fixed to the plate. After this the bellows must be 
worked more gently. Throughout the whole time care 
must be taken to brush off the frozen vapor which in a 



64 MEDICAL MICROSCOPY. 

moist atmosphere may collect below the plate^ and if not 
removed will interfere considerably with the freezing 
action of the ether, so much so, indeed, that the specimen 
may become loosened. If the ether should tend to collect 
in drops below the plate, the bellows must be worked 
more slowly. Occasionally the ether points become 
choked ; they must then be cleared by means of a piece of 
fine wire. 

When the object is completely frozen, the left hand is 
used to turn the milled head, whilst the right hand grasps 
the plane-like knife. The plate is first lowered as far as 
possible, and then the knife, at about an angle of forty-five 
degrees, is passed slowly over it, so as to cut off any por- 
tion of the specimens projecting above the level of the 
glass slide. The micrometer screw is then slightly turned 
so as to raise the specimen, and the knife is again passed 
across it. A section will thus be cut. Practice alone can 
regulate the thickness of the section. In his latest instru- 
ments Mr. Cathcart has so arranged that when the screw 
is turned through the eighth of an inch, the plate rises one 
two-thousandths of an inch. 

The above operation takes a long time to describe, but 
only a few moments to perform. By rapidly repeating it, 
some dozens of excellent sections may be obtained in the 
course of a few minutes. 

Several sections may be allowed to collect on the knife 
before removing them. They should be detached by gently 
sweeping the finger across the knife, or by means of a 
camel's hair brush, and should be immediately transferred 
to a shallow dish containing water, when by cautiously 
manipulating them with a needle they are easily separated 
from one another. 

Before again proceeding to use the knife a few puffs should 
be given to the spray apparatus to insure the mass being 



CUTTING SECTIONS. 65 

sufficiently frozen. It is sometimes advised that the knife 
should be moistened with water, but this is unnecessary, as 
the thawing of the specimen yields quite sufficient moisture 
to prevent the sections breaking. 

The knife should not be carried straight across the 
specimen, but rather obliquely, so as to expose a greater 
portion of its edge for cutting purposes, and it should be 
passed quickly and evenly along the guides. After use the 
knife must be dried with a soft cloth and passed a few 
times over a sharpening stone, especial care being used 
to remove all notches and secure a straight, sharp, and 
smooth edge. 

It is almost needless to add that the microtome must be - 
firmly clamped to the table and so arranged that the plate 
is perfectly horizontal. 

Cutting by Swift's Microtome. — The preliminary 
operation of freezing is carried out in precisely the same 
manner as just described. The cutting apparatus, how- 
ever, is quite different. It consists of a tripod stand 
supporting a razor, and is known as a ^^ plough." A 
minute description of this instrument has already been 
given in Chapter I. When arranging it for use care must 
be taken to see that the two posterior screws are on the 
same level, and the edge of the razor must be raised or 
lowered by means of the front screw until its edge is on a 
level with the top of the specimen. Thus, whereas in 
Cathcart's microtome the specimen is raised to meet the 
razor, in Swift's instrument the razor is lowered to meet 
the specimen. 

When the object is sufficiently frozen^ the plough is 
grasped with the two hands, the forefinger of the right 
being extended so as to turn the large head of the front 
screw. The razor is then passed obliquely across the 
specimen and then drawn back again, the three supports 



66 MEDICAL MICROSCOPY. 

of the tripod being firmly pressed on the glass plate. 
The front screw is next slighly turned and the process 
repeated, the thickness of the sections being thus regu- 
lated by means of this micrometer screw. When five or 
six have been cut they are cast into water in the same way 
as has been previously described. 

It is a good plan to moisten the glass plate of the 
microtome with a little glycerine, so as to enable the 
tripod to run easily. After use the knife and the entire 
apparatus should be carefully dried. 

The Use of Williams' Ice-freezing Micro- 
tome. — This instrument is too cumbersome for ordinary 
use. It takes a long time to prepare, and has no advantage 
over the two already described, except when a very large 
number of sections have to be prepared for class teaching. 

The ice-box is filled with salt and ice in alternate layers 
until the box is full. The lid is then fastened on and one 
of the discs screwed into place. The specimen is removed 
from the gum and placed on the disc, a little excess of the 
embedding fluid being added. A few minutes are allowed 
to elapse in order that the gum may freeze, and sections 
are cut with Swift's plough in precisely the same manner 
as when using his microtome. 

Cutting Sections Embedded in Celloidin. — 
For this purpose any of the microtomes may be used. 
If Cathcart's be employed, the disc must be removed and 
the clamp fixed in its place. The same if Swift's be 
chosen. It is possible to combine the freezing and 
celloidin methods; this is accomplished by cutting the 
specimens away from the corks upon which they have 
been fixed (see Chapter III), and placing them on a disc 
of a Cathcart's or Swift's microtome, surrounding them 
with water, and fixing them by freezing. 

The results, however, are only fairly satisfactory, and 



CUTTING SECTIONS. 67 

the process cannot altogether be recommended. The 
chief drawback is that alcohol cannot be used for soften- 
ing the embedding material, and consequently the sections 
are extremely apt to break and curl up. 

The best instrument to use, however, for cutting speci- 
mens embedded in celloidin is Reichert's microtome. 
Owing to the steadiness of the stand and the mechanical 
arrangement of the razor, most excellent sections can be 
obtained by its employment. The objects, having been 
duly hardened, embedded, and fixed on corks as described 
in the previous chapter, are kept in the eighty per cent, 
alcohol until required. One of them is now removed 
and the cork fixed in the clamp of the machine, and so - 
arranged by means of the screws that its upper surface is 
just below the level of the razor. The angle at which the 
specimen is set can only be determined by experiment, 
but it is a very important point; if the shape of the 
object is triangular, it is preferable that the razor should 
first encounter its largest side ; if it is quadrilateral, one 
of the angles should be placed innermost, but any project- 
ing corner should always point outward, otherwise a com- 
plete section will rarely be obtained. 

The specimen having been satisfactorily set in position, 
the next step is to arrange the knife. The rule is to allow 
as much of the cutting edge of the knife as possible to 
come into action, that is to say, the razor should be set 
very obliquely, so that it may be drawn across the tissue 
from heel to point. 

Before using the razor, care must be taken to see that 
its edge is perfectly smooth and sharp, and also that its 
carriage works easily and smoothly. 

Probably the surface of the specimen will at first be 
uneven, and will not occupy the whole extent of the tissue. 
A few preliminary cuts must therefore first be made, so as to 



6S MEDICAL MICROSCOPY. 

expose as large a surface as possible. The specimen may 
be raised between each cut in two ways, either by moving 
the micrometer screw with the finger or by the automatic 
apparatus, by means of which the screw is turned by a 
stroke of the razor at the termination of each cut. If the 
specimen be an exceptionally good one as regards consist- 
ency and uniformity of structure, the self-regulating 
arrangement is an excellent one, but this, unfortunately, is 
seldom the case, and it is therefore better to practice the 
first method, so that the thickness of each section can be 
accurately regulated. 

If sections can be obtained when three or four notches 
of the wheel have been turned, these being indicated by 
audible *' clicks," the operator may be assured that he 
has succeeded in cutting beautifully thin sections ; if five 
or six clicks have to be given before a complete section 
results, it will be found thin enough for ordinary work, 
but beyond this thick specimens only will be made, and 
some misfortune (probably unavoidable) has occurred 
in the previous treatment of the tissue, either it is of too 
diverse a structure to yield large sections (e.g., eyes or 
embryos), or it was not placed early enough in the hard- 
ening fluid, so that decomposition had set in ; or the mor- 
bid change is of such a nature that it is extremely difiicult 
to obtain sections at all, such as often happens with speci- 
mens of myelitis, or with caseous material in various 
organs. The correction of some of these difficulties will 
be considered immediately. 

In cutting celloidin specimens it is of the utmost import- 
ance that the knife and tissue should be kept covered with 
dilute alcohol (50 per cent.). The spirit should be placed 
in a capsule immediately beneath the razor, and by means 
of a camel' s-hair brush frequently swilled over the razor 
and celloidin ; if this be not done, the specimens will tend 



CUTTING SECTIONS. 69 

to break or crumble before the knife, or curl up in such a 
manner that it will be found almost impossible to unroll 
them. 

Sometimes, however, in spite of all these precautions, 
it will be found very difficult to cut delicate specimens, 
and various artifices have to be adopted. 

In the first place, sections may sometimes be obtained 
when other efforts have failed, by drawing the knife very 
slowly over the specimen, at the same time gently unfold- 
ing the section by means of the brush, and spreading it 
out on the razor. Changing the direction of the speci- 
men by removing the cork from the clamp and turning it 
round, so that another part of the tissue comes first into 
contact with the knife, will occasionally produce the 
desired result. But the most successful plan is to employ 
what is sometimes known as ** coUodionization of the 
sections/' 

This is accomplished as follows {Quart. Journ. Roy, 
Mic. Soc, 1885, P- 73^) • Some very fluid collodion is 
placed in a small bottle; it is convenient to have a bottle 
with a perforated cork, through which passes a camel-hair 
brush, or one with a glass stopper to which a small rod is 
attached. The collodion must be rendered so thin with 
ether that when applied to the surface of the celloidin it 
dries in a few seconds, and must be further diluted as soon 
as it begins to leave a shiny surface on the tissue. 

In dealing with very brittle objects, the moistened 
brush or rod (it must be no more than merely moist) is 
passed quickly over the surface of the tissue, so as to leave 
a thin film ; as soon as this is dry the section is cut. This 
method is especially useful in dealing with the central 
nervous system. It is not well to allow too many sections to 
accumulate on the knife, but after a few have been cut they 
should be transferred by means of a camel-hair brush to a 



70 MEDICAL MICROSCOPY. 

shallow dish containing water ; owing to^the rapid separa- 
tion of the spirit, they immediately spread out, and then 
may be transferred to dilute spirit preparatory to mounting. 

When the sections are very fragile, it is better to 
place dilute spirit (fifty per cent.) in the dish instead of 
water, otherwise they are very often torn in pieces by the 
rapid way in which the mixing of the spirit and water 
causes them to unfold. 

Cutting Sections Embedded in Paraffin. — The 
paraffin blocks containing the objects are pared so as to 
fit the clamp, which is arranged in the same manner as 
already described for the celloidin specimens. The suc- 
ceeding processes, however, are quite different. The 
sections must be cut dry, that is to say, no fluid must 
be used to moisten the knife. The razor also should be set 
as square as possible, for if the oblique position be adopted 
there is a great tendency for the sections to curl up. 
Should this accident occur they may sometimes be made 
to unroll by placing them in water at a temperature of 40*^ 
C. After they are cut the sections should be placed in 
turpentine or xylol, in order to remove the paraiffin. This 
takes some time, but after it is successfully accomplished 
they must be passed through absolute alcohol and weak 
spirit to water before commencing the ordinary methods 
of staining and mounting. 

Cutting in Series. — For medical work, the process of 
cutting a number of specimens in succession, so that a 
whole series may be preserved, is almost limited to the 
examination of the central nervous system and the patho- 
logical examination of the eyeball. In the physiological 
laboratory it is more commonly employed. 

Undoubtedly the most beautiful and complete series can 
be obtained by means of the Cambridge rocking micro- 
tome, but for ordinary purposes a very good series can be 



CUTTING SECTIONS. 7 1 

prepared by Weigert's method with an ordinary micro- 
tome; this is carried out as follows : — 

The specimens are best embedded in celloidin. Sections 
are cut in the usual manner, but only one must be on the 
knife at a time, and should be carefully spread out with a 
camel's-hair brush. A thin strip of tissue paper a little 
wider than the section is laid upon it, to which it adheres, 
and may thus be removed. In this manner each section 
is taken up in the order it is cut, as many strips being 
made as is necessary for completion of the series. Each 
strip should not be more than three inches long, and when 
filled should be laid on filter paper moistened with dilute 
spirit, so as to keep it damp. Glass slides are now coated 
with a thin layer of collodion and allowed to dry. On 
these the strips of paper are placed prepared face down- 
ward, so that the sections come in contact wath the col- 
lodion. A little pressure with dry filter paper will cause 
them to adhere firmly, so that the paper may be removed. 
Some more filter paper is then gently pressed over the 
sections to dry them, and finally a second layer of thin 
collodion is painted over them, and they are thus pre- 
served, sandwach wise, between the two layers. When the 
slides are immersed subsequently in the various staining 
fluids the double film is usually set free, carrying the sec- 
tions with it. Each film can then be treated as a section, 
and after the staining process is finished may be cleared 
in xylol and again fixed to a glass slide by means of 
collodion. Instead of a cover glass, a layer of photog- 
rapher's varnish may be applied. In the preliminary 
stages, instead of painting the collodion on to the slides 
with a brush, it is better to pour a little collodion on the 
slide, and then by means of tilting, a thin uniform layer 
may be obtained in the same way as in the preparation of 
photographic plates. 



72 MEDICAL MICROSCOPY. 

Cutting with the Rocking Microtome. — The 
object will probably have been embedded in paraffin ; if 
celloidin has been employed, the specimen must be removed 
from the cork, and after the edges have been carefully pared 
so as to just expose its surface, it is immersed for a few 
minutes in benzol, and then covered with paraffin wax by 
the method described on p. 56. 

The specimen is fused on to the paraffin contained in 
the socket of the microtome by means of a heated knife, 
being afterward trimmed, so that a narrow border extends 
beyond the object in all directions. 

Dr. Lovell Gulland (^Journal of Anatomy and Physi- 
ology, vol. XXV, p. 56), gives many useful hints on this 
subject. He lays stress on the importance of making the 
surface which meets the razor exactly parallel to the 
opposite surface, and of the whole block being exactly 
rectangular. A thin layer of soft paraffin is next applied 
to the upper surface (the one meeting the razor) and 
to the opposite surface; this is best carried out by 
dipping these surfaces into the melted soft paraffin, and 
when this has become firm the surfaces are again trimmed 
square. 

This is done in order to prevent any curve in the 
ribbon from being accentuated by the flattening out of 
the sections. 

The specimen mounted on the lever is brought near the 
razor by turning the milled head, and so raising the 
horizontal arm. The ratchet in connection with the 
handle is most conveniently arranged so as to catch 
seven teeth at each stroke, corresponding to about -5-oVir ^^ 
an inch thickness of the section. The handle of the 
microtome is worked backward and forward with the 
right hand, whilst the left manipulates the ribbon of sec- 
tions as it falls. 



CUTTING SECTIONS. 73 

Dr. Gulland advises that the ribbon should be divided 
into lengths corresponding to that of the slide in use. 
These divisions are to be seized at one end with forceps, 
and the other end is gently lowered on to the surface of 
warm water contained in a flat dish, placed just beneath 
the microtome; as the sections flatten out they will be 
found to move along the top of the water so that more 
and more of the ribbon can be lowered. When the 
flattening is complete, the slide, carefully cleaned, is 
immersed in water, and the ribbon floated into position on 
the slide with a stifl" brush, this process being repeated 
until the slide is full, the ribbons being laid side by side. 
It is then set up on end to allow the water to drain off. 
The temperature of the water must be regulated according 
to the melting point of the paraffin, but it should never be 
hot enough to melt the wax; short of this, the warmer 
the water the more rapidly and completely are the sections 
flattened. 

The sections are fixed on the slide by evaporating 
the water from its surface, preferably over a warm oven, just 
warm enough to drive away the moisture without melting 
the wax. Thus, if the paraffin melts at 52° C, the oven 
should be 50° C. 

When the water has completely evaporated, the opacity 
of the sections disappears, they look dry, and become 
much more transparent. When the fixation is complete, 
the paraflin is melted and washed off with turpentine 
or xylol. 

One of the greatest advantages of this method is the 
perfect ease and safety with which it allows sections to 
be manipulated on the slide, so that the most varied 
stains and reagents may be directly applied. 
7 



CHAPTER V. 

STAINING. 

Sections may be examined directly they are cut with- 
out undergoing the process of staining; although consid- 
erable information may be gained in this way, far more 
can be obtained when certain dyes have been employed ; 
in this way special structures are brought out which 
would otherwise have escaped notice, and the various 
tissues are differentiated one from another, owing to their 
unequal receptivity for the stain or stains. 

The chief object of staining is therefore a selective one. 
The differentiation may be accomplished either by the 
employment of one stain, which picks out certain elements 
of the tissue, either leaving the rest colorless, or by a 
chemical action producing quite a different hue. An 
example of the first class is haematoxylin, which picks out 
the nuclei, leaving the ground substance almost uncolored, 
or only of a delicate violet tint ; an instance of the 
second class is methyl aniline violet in presence of 
amyloid material ; this it stains red, the rest of the section 
assuming a violet tinge. A more common plan is to use 
two stains, one of which colors the nuclei and the other 
the ground substance ; for example, haematoxylin and 
eosin, carmine and picric acid. 

The selective power of stains is aptly classified by 
Mr. Arthur Lee ('' Microtomist's Vade Mecum ") into 
*' histological selection and cytological selection." In 
the former, an entire tissue or group of tissue-elements 

74 



STAINING. 75 

is prominently stained, the elements of other sorts pres- 
ent in the preparation remaining colorless, or being 
differently stained, as in a successful impregnation of 
nerve endings by means of gold chloride. In the latter, 
the stain seizes on one of the constituent elements of cells 
in general, either the nucleus or the extra-nuclear parts. 

Owing to the selective power of the different stains, 
they have been divided into nuclear, general, and select- 
ive, according to whether they color the nuclei only, the 
whole tissue, or particular elements, such as bacteria and 
fat (see ''Manual of Clinical and Practical Pathology," 
by Dr. Wynter and the Author). 

There are two chief methods of staining, the direct 
and the indirect. In the first, the tissue is colored by 
some dye which seizes upon one particular group of 
elements, such as the nuclei, more quickly than upon the 
other constituents of the tissue, and the process is stopped 
when the former are sufficiently colored, whilst the latter 
are merely tinged ; an instance of this class is hematoxylin. 

The indirect method, however, applies to the great 
majority of stains. In this, the tissues are first over- 
stained, and then the color again partially removed by 
some agent, certain elements retaining the color longer 
than others. An explanation of this action is difficult to 
find j in the case of bacteria, an ensheathing envelope 
may be the resisting power to the decolorizing media. 
The aniline dyes belong to this class, and most beautiful 
results are obtained by their use. 

In this chapter the use of those stains will be considered 
which are applicable to ordinary histological processes ; a 
description of selective stains, such as those suitable for 
the central nervous system and those used for bacterio- 
logical purposes, will be given in succeeding chapters. 



76 MEDICAL MICROSCOPY. 

A few words are necessary concerning the management 
of stains generally and the manipulation of sections. 

The stains should be procured as pure as possible, and 
therefore it is much better to get them from the regular 
dealers, such as Becker, Pillischer, Martindale, Allan and 
Hanbury, etc., than from any chance druggist. They are 
best preserved in smoked glass bottles. 

All stains must invariably be filtered before use, as 
deposits are very apt to occur, and unless these be 
separated, the sections will be entirely spoiled. 

For holding the stains when in use, small glass capsules 
or watch glasses, with ground under-surfaces to keep them 
steady, are usually employed, but the most convenient are 
solid blocks of glass hollowed out, such as are sometimes 
used as salt cellars. These hold about the right quantity 
of fluid, are of the right depth, very firm, and can with 
safety be covered over with small squares of glass so as to 
exclude dust. This is most important, especially when 
the sections have to remain for several hours exposed 
to the coloring agent, and is absolutely essential when 
alcoholic solutions are used. 

It will be seen that some of the dyes are in watery 
solutions, others in alcoholic ; when they have not been 
diluted, ihey may be filtered back into the bottle after 
use, for the sake of economy, but the alcoholic solutions 
have frequently to be diluted, and they cannot then be 
employed a second time. 

In the prececfing chapter, after the sections were cut, 
they were directed to be kept in dilute methylated spirit 
(equal quantities of spirit and water) until wanted. If 
the dye has been dissolved in spirit, the sections may 
be transferred directly to the stain, but if a watery solution 
be employed, they must be rinsed for some minutes in 



STAINING. 77 

water until the alcohol is removed, and with delicate 
sections care must be taken that the rapid separation of the 
spirit does not cause them to fly into pieces. 

A section is most conveniently transferred from one 
solution to another on the end of a glass rod drawn out to 
a fine point ; a gentle movement of the rod will dislodge the 
section, and with a little practice it may be easily made to 
spread out. In the early stages it is not essential that the 
section should be kept flat, but it is advisable to do so 
if possible. Glass rods are to be preferred to needles, as 
they are less liable to catch in the section and tear it. 
The advice sometimes given to always move sections 
by means of a section-lifter is not altogether good, as 
a certain amount of pulling of the section is then required, 
and a delicate one will very likely be considerably damaged 
thereby. Special directions will be given for the manipu- 
lation of fragile specimens. 

The Choice of a Stain. — In preparing ordinary 
sections (as distinguished more especially from bacterio- 
logical workj there are two combinations which are most 
commonly employed, namely, haematoxylin and eosin, 
and carmine and picric acid ; they yield excellent results, 
and the student cannot do better than employ them. A 
great number of other double and treble stains are con- 
stantly being suggested, but these two have stood the test 
of time. They are simple to carry out, not requiring 
a complicated series of changes from one reagent to 
another. 

These methods will first be described. 

Staining with Haematoxylin and Eosin. — Sections 
stain best with haematoxylin which have been hardened 
in alcohol and Miiller's fluid, but Delafield's preparation 
will also stain preparations which have been hardened in 
chromic or osmic acid. 



78 MEDICAL MICROSCOPY. 

The best formula is that which was originally introduced 
by Delafield. It is made as follows : — 

To I GO c.c. of a saturated solution of ammonia alum, 
a solution of one gramme of haematoxylin, dissolved in six 
c.c. of absolute alcohol, is added, drop by drop. The solu- 
tion is exposed to the air and light, in an unstoppered 
bottle, for three or four days ; it is then filtered, and 
twenty-five c.c. of glycerine and twenty-five c.c. of 
methylic alcohol are added. The mixture is allowed to 
stand until its color becomes dark, and is then again 
filtered and preserved in a bottle with a closely fitting 
stopper. It keeps well, but should not be used for two 
months after it has been prepared. 

Another formula for making the stain is the one that 
bears Ehrlich's name. It can also be highly recommended, 
but it has no advantage over Delafield's, and will not stain 
specimens which have been hardened in chromic acid. 
Its composition is as follows : — 

Haematoxylin, 2 parts, 

Alcohol, lOO *' 

Distilled water, loo " 

Glycerine, loo *' 

Alum, 2 ** 

Glacial acetic acid, . . . . _ 4 *' 

Dissolve the haematoxylin in the alcohol, add the 
glycerine and water, and finally the acetic acid. The 
solution will not have acquired its full staining powers 
until a week after it has been made. The mode of using 
the above solutions is the same. 

There are two methods of staining by haematoxylin, a 
rapid and a slow. In the rapid process the sections are 
dipped for about half a minute into the undiluted stain 
and are then thoroughly washed in water. Specimens col- 
ored in this way are not always satisfactory j they are 



STAINING. 79 

likely to be stained unequally, being over-stained in one 
pjart and not sufficiently so in another. 

The slow method yields far more satisfactory results. 
Distilled water is placed in a capsule until it is about half 
full, a few drops of the stain are then added, and the 
whole filtered. The more dilute the mixture the longer 
must the sections remain in it. A convenient plan is to 
add about half a dozen drops of the haematoxylin solution, 
and then allow the specimens to remain in the mixture all 
night. They are then removed and thoroughly washed in 
water ; a stay of two hours or more in the water greatly 
improves the color. Afterward one of the sections is 
placed on a glass slide, spread out and placed under the 
microscope, and examined by an inch objective. It will 
then be seen whether it is properly colored. If it be not 
sufficiently so, the spejcimens must be put back in the 
stain, but if they are too dark they may be washed for a 
few seconds in a five per cent, solution of acetic acid. 
Overstaining is to be avoided, if possible, as after the 
action of the acid they do not keep their color well, and 
soon become useless. 

After being properly stained in haematoxylin, a good 
effect is produced by ^* counter-staining " them in eosin. 

For this purpose the following solution must be 
made : — 

Eosin, 5 parts, 

Distilled water, loo ** 

The sections are removed from the water in which they 
have been washed, and allowed to remain for about five 
minutes in the stain. They are then swilled in water and 
rapidly dehydrated in absolute alcohol, cleared in clove 
oil, and mounted in balsam, as will be described in the 
chapter devoted to *' mounting." 



8o MEDICAL MICROSCOPY. 

It must be stated here that the eosin is quickly dis- 
solved out of the sections by absolute alcohol and oil of 
cloves, and therefore these processes must be performed as 
rapidly as possible. It is a good plan to add a few drops 
of eosin to the alcohol. 

Thus prepared, the nuclei are stained a deep bluish- 
purple, those of the epithelial cells being less darkly colored 
than those of the lymphoid cells and connective tissue. 
The ground substance assumes a clear rose-color, the 
fibrous tissue being especially picked out. This method 
is particularly applicable to specimens of cirrhosis. 

Double staining by haematoxlyin and eosin may be 
accomplished in one process, by using Renaut's haematox- 
ylic eosin.'' It is made as follows: — 

Concentrated watery solution of eosin, .... 30 parts, 
Saturated solution of potash alum in glycerine, . 130 " 
Saturated solution of hsematoxylin in alcohol, . 40 " 

The eosin solution is added, drop by drop, to the potash, 
and the mixture filtered ; then the haematoxylin solution is 
also added, drop by drop, and the mixture set aside for five 
or six weeks in a vessel with a perforated cover, until the 
alcohol is evaporated, when, after filtering, the stain is 
ready for use. 

The best method for using this stain is to mount the 
objects in the fluid diluted with one or two volumes of 
gylcerine, as the color is very slowly absorbed. After a 
few weeks the specimen will have becomxC perfectly clear. 
The cover glass should be fixed with balsam or gum 
damar. 

This stain has a selective action on the cells of the 
salivary mucous glands; mucous cells it colors a pale 
blue, whilst the demilunes become a deep rose color. 

Staining by Carmine and Picric Acid. — A very 



STAINING. 8l 

large number of preparations of carmine are used for 
microscopic purposes. As with haematoxylin and eosin, a 
simple method will first be given in its entirety, and then 
the other preparations of carmine will be briefly con- 
sidered. 

Carmine is a most excellent nuclear stain. It is rather 
more trying to the eyes than haematoxylin, but is more 
permanent. The usual method for its use is, first to 
stain the specimens throughout, and then wash out the ex- 
cess of color from everything except the nuclei. But if 
no such reagent be employed the carmine will be found to 
stain the nuclei a bright red, and in addition to color, 
though not so brilliantly, the fibrillar ground substance of 
the connective tissue, muscular fibres, the ground substance 
of bony tissues, fibrine, the neuroglia of the central 
nervous system, the axis cylinders of nerves, etc., leaving 
uncolored elastic tissue, the white substance of Schwann 
in the nerves, fat, mucous substances, chalky deposits, 
etc. 

The most convenient formula is lithium carmine. 
This is very largely used in the laboratories of Vienna, 
but not so well known in England. It produces very 
good results, and its manipulation is easy to learn. It is 
made as follows: — 

Carmine, 2.5 parts, 

Saturated solution of lithium carbonate, , . 100 " 

The mixture requires to stand for some time and to be 
carefully filtered before use. 

Sections are allowed to remain in the undiluted stain 
for about five minutes. A longer stay will not be harm- 
ful, as over-staining is not easily accomplished, in fact, if 
the sections remain for twelve or twenty-four hours in the 
dye they will be found to be by no means spoiled. 
. 8 



S2 MEDICAL MICROSCOPY. 

After -removal from the stain the sections are at once 
placed in acidulated alcohol : — 

Hydrochloric acid, I part, 

Alcohol, 70 parts, 

Distilled water, 30 '* 

The specimens will be at once seen to give up a great 
deal of color. They should occasionally be slightly 
moved about so as to distribute the cloud which forms 
around them. Two or three minutes usually suffice for 
decolorization ; but they should not be removed from the 
solution so long as any color comes out. It is often neces- 
sary to have two basins containing the dilute acid, so as 
to ensure all the superfluous color being removed. They 
are next transferred to absolute alcohol to which five or 
six drops of a concentrated alcoholic solution of picric acid 
have been added. This dehydrates the sections, as well as 
counter-staining them, and after two minutes they may be 
removed to oil of cloves, or creosote, preparatory to 
mounting in balsam. As both these oils remove the picric 
acid rapidly, a few drops of the concentrated solution of 
picric acid should be added to the clearing reagent. 

Staining with carmine and picric acid may be accom- 
plished in one operation by using the stain known as 
*' picro-carmine." Sections colored in this manner 
cannot be mounted in balsam. The solution is rather 
difficult to prepare, but the directions given by Dr. Sims 
Woodhead (*' Practical Pachology," p. 56) are thoroughly 
reliable, and the observer cannot do better than follow 
them closely : — 

'' Take of— 

Pure carmine, i part, 

Strong ammonia, 3 parts, 

Distilled water, 3 " 



STAINING. 83 

*' Dissolve the carmine in a test-tube with the ammonia 
and water. To this add 200 parts of a cold, saturated, 
and filtered solution of picric acid, and mix thoroughly. 
Place the fluid in a basin and cover with a clock glass 
(with the concave surface upward to keep out dust, and to 
allow of the moisture falling back into the basin, so that 
the exposure to the sunlight may be prolonged), and allow 
it to evaporate in strong sunlight, testing it every few days 
by staining sections in it, until the nuclei and fibrous tis- 
sue are stained distinctly pink, whilst epithelial cells, etc., 
are stained yellow. The best double staining is usually 
given before the fluid has evaporated down to half its bulk, 
and at this stage it is sometimes found that crystals of 
picric acid are deposited in the tissues. To obviate this, 
it is necessary to add 10 or 20 parts of distilled water. To 
prevent the growth of fungi, add from 2 to 6 drops of i 
in 20 carbolic acid solution to each ounce of the fluid ; 
filter and keep in a glass-stoppered bottle.'' 

Sections are stained in the following manner : Alcohol 
must first be abstracted by allowing the specimens to float 
in water for some minutes. They are then removed one 
by one to glass slides and carefully spread out. A few 
drops of the staining fluid are added to each and allowed 
to stand for about five minutes. The excess of stain round 
the sections is then wiped ofl* with a cloth, but the sections 
themselves are not dried. A little Farrant's solution is 
then added, cover-glasses applied and fixed by ^^ ringing'' 
with Dammar varnish or balsam. 

If examined immediately the sections will not appear to 
be properly colored, but this result will be satisfactorily 
completed in a few days, when the excess of staining fluid 
will be found to have become absorbed, and a most beauti- 
ful selective double staining will be brought about. 

W€ now pass to a short consideration of the other car- 



84 MEDICAL MICROSCOPY. 

mine stains which are employed. These are given rather 
for the sake of completeness than for any advantage which 
they possess over lithium carmine. An exception must be 
made in the case of ammonium carmine, which is especi- 
ally valuable for staining sections of the central nervous 
system. 

Alum Carmine. — 

Carmine, I part, 

Alum (5 per cent, solution), loo parts. 

The carmine is added to the alum, boiled for a quarter 
of an hour, and then filtered. A little carbolic acid 
should be added for preservation. 

Sections are allowed to remain in the undiluted stain for 
about ten minutes. They are then washed in acidulated 
alcohol (see p. 82) until no more color comes out, dehy- 
drated, cleared, and mounted in balsam. 

Ammonium Carmine. — 

Carmine, I part. 

Strong ammonia, I '* 

Water, 100 parts. 

Place the carmine in a mortar and add sufficient water 
to make a paste ; then pour in the ammonia, filter, and 
place a small piece of camphor in the bottle for preserva- 
tion. 

As already stated, this stain is especially applicable for 
sections of the central nejvous system. 

After removal from the hardening reagent (chromates) 
the specimens should be dipped for about ten minutes in a 
.2 per cent, solution of chloride of palladium. 

Sections must remain in the carmine for about ten 
minutes, and be subsequently washed in water. 

The medullary sheaths of the nerves are stained yellow, 



STAINING. 85 

the nuclei, ganglion cells, and axis- cylinders being colored 
a deep red tone. 

Friedlander states that this dye is well adapted for 
staining fresh bone specimens which have not been 
artificially decalcified. It is thus very useful in specimens 
of rickets and osteomalacia. 

Borax Carmine (Grenacher). — 

Carmine, 5 part, 

Borax, 2 parts, 

Water, loo '* 

The mixture is placed in a porcelain dish and heated to 
boiling, and a 5 per cent, solution of acetic acid is added ' 
until the purple color changes to red. The solution is 
allowed to stand for twenty-four hours, filtered, and pre- 
served with a few drops of carbolic acid. 

Sections are stained in a few minutes, and are subse- 
quently partially decolorized in acidulated alcohol. The 
stain is useful for sections of the spinal cord and brain. 
Its chief use is for staining specimens in ** bulk," that is, 
to stain preparations after they have been hardened and 
before sections have been cut. 

This method is generally used for portions of the spinal 
cord and brain. They are allowed to remain in the undi- 
luted stain for from twelve to forty-eight hours, according 
to the size of the specimens, and then from three to twelve 
hours in the acidulated alcohol, until no more color can 
be extracted. They are next transferred to pure methy- 
lated spirit and absolute alcohol in succession, remaining 
in the last named not less than three hours, after which 
they are removed to about ten times their bulk of chloro- 
form or benzol until thoroughly saturated, and are finally 
embedded in paraffin in the usual way (see p. 60). Sec- 



S6 MEDICAL MICROSCOPY. 

tions are cut, and after dissolving out the paraffin they 
are cleared and mounted in balsam. 

Two other nuclear stains may be referred to here, 
namely, safranin and iodine green. They do not present 
any great advantages over haematoxylin and carmine, but 
may be used for the sake of variety. 

Safranin is one of the best nuclear stains we possess, 
and is therefore especially useful in studying rapid cell- 
formation in morbid growths, or the process of karyoki- 
nesis. It is also an excellent stain for epithelial structures. 
It communicates a brilliant orange-red color to nuclei. 
Its powers as a selective stain for colloid material will be 
again referred to in a future chapter. 

In preparing the stain great care must be taken to 
procure a good sample ; a reliable dye may be obtained 
from Dr. George Grlibler, Baiersche Strasse 12, Leipsig. 

The best formula is : — 

Concentrated alcoholic solution of safranin, ... I part, 
Concentrated aqueous solution of safranin, ... I " 

Sections are allowed to remain in the stain for about a 
quarter of an hour, and are then washed in absolute 
alcohol until they become almost colorless, when they are 
removed to oil of turpentine for the purpose of clearing, 
and may then be mounted in balsam. 

Iodine Green. — This stain is best used in the form ot 
a one per cent, aqueous solution. Sections placed in 
it stain almost instantaneously, and are improved by pre-" 
vious floating in water. After staining they are washed 
out in water, dehydrated in alcohol, and mounted in 
balsam. 

With ordinary tissues it has peculiar differentiating 
powers, producing three shades of color in those elements 



STAINING. 87 

which it selects. Thus epithelial cells and nuclei are 
stained dark blue ; glandular structures dark green, whilst 
muscular fibres acquire a malachite green tinge. This dye 
has also a selective action for amyloid material, which it 
colors a rose-pink. 

In order to produce a contrast in the parts of the tissue 
other than the nuclei, certain general stains are used 
with the above nuclear stains. Eosin and picric acid as 
contrasts to haematoxylin and carmine respectively have 
already been referred to. Eosin is also much used in the 
examination of blood, as will be explained in the chapter 
devoted to that subject. 

A few other general stains will be mentioned here, which - 
the reader may use at his pleasure. 

Bismarck Brown. — This dye, also known as ** vesu- 
vin," is more used as a contrast stain in bacteriological 
work than with ordinary microscopic specimens. It is, 
however, very valuable for staining sections of bone and 
those exhibiting young granulation tissue. 

The most convenient form in which to use it is : — 

Bismarck brown, 2 parts, 

Alcohol, 15 " 

Distilled water, 85 " 

It yields a light oak tint, which is very generally dis- 
tributed over the mass of the tissue. 

The sections should be allowed to remain in the stain 
for about a quarter of an hour, and then be swilled in 
water before dehydrating. 

This dye forms a pleasing contrast stain to safranin or 
haematoxylin. 

Rubin. — A 5 per cent, watery solution is the best 
strength to use. Sections are allowed to remain in the 
stain for about five minutes, are then washed in water and 



SS MEDICAL MICROSCOPY. 

transferred to absolute alcohol ; a good deal of the color 
will now be taken out, and this should be allowed to con- 
tinue until the specimens are light pink in color, when 
they should be removed to oil of cloves and finally 
mounted in balsam. 

Rubin colors the tissues a claret-red and thus presents 
a good contrast to haematoxylin ; like eosin^ it picks out 
the fibrous tissue very beautifully. It is an excellent stain 
for the clubs of actinomyces (see p. io6). 

Orseille. — The stain is made thus ; — 

Absolute alcohol, » . . . 20 parts, 

Acetic acid, 5 " 

Distilled water, 40 " 

Pure orseille is then added in sufficient quantity to pro- 
duce a dark-red liquid. 

The solution must be filtered before use. This dye is 
but little used except for staining the clubs of actinomyces, 
being introduced for this purpose by P. Israel. It is a 
fair contrast stain for haematoxylin, coloring the general 
tissue a deep crimson. Sections require to be left in the 
stain for about ten minutes, and are then washed in water, 
cleared, and mounted in balsam. 

Multiple Staining. — The majority of sections which 
are now prepared in the laboratories are double-stained, 
as by this means the several parts of the tissue are demon- 
strated more clearly than when only a single stain is used. 
The two most common and most satisfactory combinations, 
namely, haematoxylin and eosin, and carmine and picric 
acid, have already been considered. Other arrangements 
of dyes may be employed, according to the fancy of the 
worker, and the method of combination can easily be in- 
vented by referring to the dyes under the various heads of 
** nuclear '^ and '^ general " stains. 



STAINING. 89 

In Lee's ^* Microtomist's Vade Mecum," the reader will 
find a large variety of combinations, the best amongst 
which are : — 

Picro-carmine and iodine green. 

Methylene blue and eosin. 

Methyl green and eosin. 

Methylene blue and Bismarck brown. 

Fuchsine and methylene blue. 

Treble Staining. — The use of three colors is hardly 
of any advantage ; the process becomes tedious, and no 
fresh information can be derived from the addition of a 
third dye. To those who wish to make the experiment, 
we recommend Dr. Heneage Gibbes' process (slightly 
modified) as given in his work on '^Practical Pathology 
and Morbid Histology," p. 51, as being the most satisfac- 
tory yet introduced. 

The stains required are : — 

Solution I. — Lithium carmine (see p. 81.) 

Solution 2. — Rosanilin (Gibbes' formula, opus cit., 

p. 47)- 

'^ Place some of the crystals of rosanilin hydrochloride in 
a glass mortar and rub up with a little spirit ; add more 
spirit until all the crystals are dissolved." 
Solution ^. — Iodine green (see p. '^(y). 

The method is carried out as follows : — 

The sections are first stained in lithium carmine accord- 
ing to the process already described, and are then soaked 
in acidulated alcohol. A few drops of the solution of 
hydrochloride of rosanilin are then diluted with spirit, the 
sections immersed for two or three minutes, and subse- 
quently removed to methylated spirit to wash off the ex- 
cess of coloring matter. They are next placed in a dilute 
aqueous solution of iodine green. 

Coming from spirit, they float on top of the watery solu- 



90 MEDICAL MICROSCOPY. 

tions, and this in many cases, when the green stain is not 
required to be very deep, is quite sufficient. When a 
deeper stain is required, they must be immersed altogether, 
and allowed to remain for a minute or two, but it must be 
borne in mind that this color cannot be washed out again 
if too deep, while the spirituous stain can, so that it is better 
to have a section apparently over-stained in the rosanilin 
solution, while it is even under-stained in the iodine green. 
After washing, the sections are dehydrated, cleared, and 
mounted in the usual manner. It will be found, however, 
that a good deal of the rosanilin will come out in the 
second immersion in spirit, and it is necessary to change 
it until no more color comes away, otherwise the oil of 
cloves will become colored, and from it the Canada bal- 
sam in which the specimen is mounted. 

Dr. Gibbes adds: *^With the above-mentioned three 
colors, the most beautiful effect may be obtained, but it 
will take some time and practice to get the process exactly 
right, and proficiency in the matter can only be gained by 
experience. The results will be found to vary with the 
length of time the section is immersed in each of the two 
last colors, and also with the strength of the solutions. 
The best results will also be obtained with material that has 
been hardened in chromic acid." 

If sections of tongue are stained by this method, all the 
muscular fibres will be stained with the carmine, as also 
will the connective tissue, protoplasm of cells, etc., while 
all the nuclei in the superficial epithelium, serous glands, 
non-striped muscle tissue in the vessels and elsewhere are 
stained a brilliant green. 

The most important fact demonstrated by this process 
is the different reactions shown by the various glands. In 
the mucous glands, although the epithelium lining the 
duct is stained in the same manner as the superficial 



STAINING. 91 

epithelium of the organ, yet the secreting epithelium 
assumes anew color, differing entirely from either of those 
employed, namely, a sort of purple. In the serous glands, 
however, there is no such change ; the protoplasm of the 
cells is stained more or less deeply with red, while the 
nuclei have taken on a green color. 



CHAPTER VI. 

SELECTIVE STAINS. 

Under the term ^^ selective stains'' we include those 
dyes which, for special reasons, pick out various com- 
ponents of the tissues (other than the nuclei) or bodies 
contained in them (micro-organisms). This selective 
power is exerted in some instances by a species of chemi- 
cal reaction, such as the precipitation of reduced metal 
in the presence of light, as is the case with gold, silver, 
and osmium salts; or in another class of cases the 
singular production of color by amyloid material. In 
other instances the selective action is dae to the power of 
micro-organisms to retain dyes which are removed by 
suitable agents from the other parts of the tissue. Of this 
kind are the large and valuable series of aniline dyes. 
Since these have been introduced into microscopical 
technique an immense advance has been made in our 
knowledge of bacteria. If a section be placed in a solution 
of one of these dyes for about five minutes and then re- 
moved and placed in water, it will be found to be opaque, 
and colored almost black, whilst at the same time a 
peculiar shimmering cloud is formed around it. But if 
the section be removed to alcohol, a large amount of color 
is at once given up, the specimen gradually becomes paler, 
more transparent, and assumes a violet, blue, or reddish 
tinge (according to the dye used). When this stage is 
reached the process must be stopped and the section re- 
moved to cedar oil for clearing. The exact time at which 

92 



SELECTIVE STAINS. 93 

this removal must be effected can only be learned by ex- 
perience. 

Some of the aniline dyes, such as methylene blue, are 
frequently used as simple nuclear stains, but possess no ad- 
vantage for this purpose over hsematoxylin or carmine. 

We will first deal with metallic stains. These are some- 
times known as '^ impregnation methods, '^ and are of two 
kinds. In a ^^ negative impregnation" the intercellular 
substances alone are colored, the cells themselves remain- 
ing colorless or very lightly tinted. In a '^positive im- 
pregnation" the cells are stained whilst the intercellular 
spaces remain unaffected. This last variety, however, is 
very seldom used. The exact nature of the deposit: 
formed in the intercellular spaces by the use of metallic 
stains is not known. Von Recklinghausen considered that 
the silver salt combined with a hypothetical intercellular 
cement substance, forming a compound that blackened 
under the influence of light. Other authors are of opinion 
that the metallic salt combines with albuminous and saline 
liquids that surround the cells, and is then precipitated in 
simple intercellular spaces. 

Nitrate of Silver. — The most convenient strength for 
using this reagent is a half per cent, solution in distilled 
water. It is more frequently employed for physiological 
than for pathological work. It is especially useful for 
pathological conditions of the eye and for tumors of epi- 
thelial type. It can only be used when the specimens are 
perfectly fresh, and its employment is therefore limited to 
structures which have been removed during life. Very 
thin sections must be made, which are washed in distilled 
water to remove chlorides, and are then placed in the 
reagent for about five or ten minutes. They soon become 
white, and as soon as a grayish appearance is noticed they 
must be washed in distilled water, and then removed to 



94 MEDICAL MICROSCOPY. 

ordinary water and exposed to the daylight until they be- 
come brown. The sections may be preserved in spirit 
until wanted or mounted at once in glycerine. Such 
specimens must be kept in the dark or they will become 
black and opaque. 

Sections may be counter-stained with lithium carmine 
and picric acid, or other carmine dyes, care being taken, 
however, to avoid solutions containing free ammonia, 
which would dissolve out the silver. During impregnation 
by the silver salt, unless the sections or membrane be kept 
perfectly flat, the silver salt will be deposited unequally. 
To prevent this it is a convenient plan to pass the mem- 
brane over a porcelain ring, and fix it with another ring in 
the manner in which a skin is fitted on to a drum (Stirling). 

A remarkable example of the positive impregnation by 
silver is exhibited by the condition known as argyria, in 
which silver salts are deposited during life in various tissues 
of the body, such as the Malpighian tufts of the kidney, in 
those patients who are taking silver by the mouth. 

Chloride of Gold. — In order to demonstrate the peri- 
pheral nerve endings, or the connective tissue corpuscles of 
the cornea, no reagent produces better results than chloride 
of gold. It is, however, extremely difficult to manipulate, 
and satisfactory specimens can only be obtained after long 
practice and with considerable exercise of patience. 

The same strength is used as for nitrate of silver, 
namely, half per cent, solution in distilled water, and 
the specimens must be perfectly fresh. A large number ot 
methods for staining by chloride of gold have been intro- 
duced. Lee's ^* Microtomist's Vade Mecum " mentions 
no less than sixteen, but only the two yielding the best 
results will be described here, namely, Cohnheim's method, 
and Ranvier's lemon-juice method. 

Cohnheim^ s Method, — Small portions of the tissue imme- 



SELECTIVE STAINS. 95 

diately after removal from the body are placed in the chlo- 
ride of gold solution of the above strength until they are 
thoroughly yellow. They are then removed to water acidu- 
lated with acetic acid, until a purplish tinge is assumed, by 
which time the gold is reduced. About four days are re- 
quired for this process. Sections are cut and mounted in 
glycerine. Specimens thus prepared will only keep a short 
time. 

Ranvier s Lemon-juice Method. — This is especially useful 
for demonstrating nerve endings. Portions of perfectly 
fresh tissue are soaked in lemon juice, filtered through 
clean, starchless muslin until they become transparent. 
This will require from five to ten minutes. They are then' 
rapidly washed in water, and placed for about twenty min- 
utes into one per cent, gold chloride solution. After 
which they are again swilled in water and then immersed 
in a bottle containing one per cent* of acetic acid in dis- 
tilled water. 

During this period they must be carefully kept in dark- 
ness. They are then exposed to the light for about twenty- 
four hours, after which sections may be cut by freezing and 
mounted in glycerine. Specimens thus prepared are not 
permanent. In order to procure such, after staining in 
the gold and washing in water, the specimens should be 
kept for twenty-four hours in a twenty per cent, solution 
of formic acid protected from light. The reduction of the 
metal is then complete, and after washing in distilled water, 
sections may be cut and mounted in glycerine. 

Osmic Acid. — The selective action of osmic acid on 
fat has already been referred to when speaking of this 
reagent as a hardening medium. It has also a selective 
property on medullated nerve fibres, which is made use of 
in the Pal-Exner method of staining the central nervous 
system. When used as a staining reagent it is kept as a 



g6 MEDICAL MICROSCOPY. 

one per cent, watery solution obtained by breaking the 
glass tube in which it is supplied and adding one hundred 
parts of distilled water. The solution is then transferred 
to a black bottle. Sections which have been previously 
soaked in some chromate solution for hardening purposes (as 
before stated, alcohol must be avoided when we wish to 
demonstrate fat) are immersed for about six hours in a solu- 
tion of one-sixth per cent, of the acid, being meanwhile care- 
fully protected from the light. They are afterward washed 
in water and mounted in Tarrant's solution. As a rule, 
such specimens are at once hardened and stained in osmic 
acid (see p. 51), and after sections have been cut they 
may be counter-stained in picro-carmine. 

We now come to consider the large subject of staining 
micro-organisms in animal tissues. During recent years no 
branch of microscopy has made greater advances than 
this ; it has, in fact, almost become a science in itself, and 
the various processes introduced are so numerous as to need 
a volume of no mean size for a description of the whole of 
them. 

Some of the methods are directed to the recognition of 
definite pathogenic organisms, such as Ziehl's and Neel- 
sen's for tubercle and leprosy bacilli; other methods, such 
as that of Gram, will stain the majority of micro-organisms, 
whilst another, namely Klihne's, will color all known bac- 
teria but two. 

Tor a complete description of the various methods, the 
reader is referred to the larger treatises on Bacteriology, 
but an effort will here be made to present in a practical 
form such of the processes as are required for staining the 
most important pathogenic micro-organisms. 

Methylene Blue. — As '^L5ffler's solution," this dye 
has long been used for staining bacilli in sections, espe- 



SELECTIVE STAINS. 97 

cially those connected with enteric fever, diphtheria, and 
glanders. This method has now been in a great measure 
superseded by that introduced by Kuhne. Loffler's is, 
however, much more simple, and yields very good results, 
and for general work is to be recommended, whilst for 
more extended study, and especially in research, Klihne's 
should be used. 

Loffler's Method.— 

Loffler's solution has the following composition : — 

Methylene blue (concentrated alcoholic solu- 
tion), 30 parts, 

Solution of potash (i to 10,000), lOO " 

The best results are obtained from this solution, but- 
bacilli can be quite well demonstrated by a more simple 
solution, prepared thus: — 

Methylene blue, 2 parts, 

Alcohol, 15 " 

Water, , . . . 85 '• 

Sections are allowed to remain in either of the above 
stains, undiluted, for at least twelve hours, a period of 
twenty-four hours being better. A dilute solution of acetic 
acid is then made by adding about three drops of the 
strong acid to a medium-sized capsule of water, or one 
drop to a watch-glass full. In this mixture the sections are 
washed after removal from the stain until they are light blue 
in color ; they are then removed to absolute alcohol, when 
still more color is removed, and afterward they are cleared 
in cedar oil and mounted in balsam. 

It requires some practice to conduct the process of wash- 
ing out the blue or '' differentiating " the specimens prop- 
erly ; usually too much color is removed both from the 
tissues and the bacilli, or too much of the dye is left in the 
tissues, so that the bacilli can hardly be distinguished. 
9 



98 MEDICAL MICROSCOPY. 

Under such circumstances the margins of cells or other 
constituents of the tissues, especially *^ mast-zellen " (see 
below), are very apt to be mistaken for micro-organisms. 

Kiihne's Method. — There are several points in which 
Kiihne^s method of staining micro-organisms with methyl- 
ene blue differ from those previously introduced, notably 
in the addition of carbolic acid to the stain, and the use 
of aniline oil for the purpose of dehydration instead of 
absolute alcohol. 

In his general remarks on this stain, Klihne states that he 
has succeeded in staining in the tissues almost all forms of 
pathogenic bacteria, the only exceptions being the bacilli 
of leprosy and m.ouse septicaemia. The bacillus of glan- 
ders also stained in far fewer numbers than in cover-glass 
preparations. He suggests that as leprosy bacilli will not 
color by this method, it might be made use of as a differ- 
ential test in doubtful cases of tuberculosis and leprosy. 

For this method the following solutions are required : — 

Solution I. — ^' Carbol. methylene blue." 

Absolute alcohol, 10 parts, 

Methylene blue, i-5 '* 

Carbolic acid (5 per cent, aqueous sol.), . . 100 " 

The dye is placed in a mortar and the alcohol poured 
over it, the carbolic acid solution being gradually added 
during stirring. 

Solution 2. — '^ Weak acidulated water." 

Hydrochloric acid, 10 drops. 

Water, 500 c. c. 

Solution 3. — '' Lithia water." 

Water, 10 c. c, 

Concentrated anueous solution of lithium ) /- ^ o j 

, ^ ' V 6 to 8 drops, 

carbonate, j ^ 

Solution 4. — ^^ Aniline oil solution of methylene blue." 



SELECTIVE STAINS. 99 

About as much methylene blue as will go upon the 
point of a pen-knife is rubbed up in a mortar with lo c. c. 
of aniline oil. The whole of this mixture (the dye will 
not be wholly dissolved) is then poured into a small bottle. 
When required for use a few drops of the solution are 
added to a small capsuleful of pure aniline oil. 

Solution 5. — ** Aniline oil solution of safranin." 

This is made in the same way as Solution 4, using safranin 
instead of methylene blue. 

Mode of procedure : — 

Sections are removed directly from alcohol into the car- 
bolic methylene blue (Solution i), where they remain for 
about half an hour. The specimens are next washed in 
water, and then differentiated by rinsing in weak acidu- 
lated alcohol (Solution 2), being afterward dipped for a 
few seconds in lithia water (Solution 3) and again im- 
mersed in ordinary water. The process of decolorization 
must be carefully watched, and stopped when the speci- 
mens have assumed a pale blue tint. The time for this varies 
from a few seconds up to a quarter of an hour or more. 
Experience alone can guide one, and many disappoint- 
ments will probably be experienced before success is 
attained. After remaining for about five minutes in water, 
the sections are dipped for a second or two into absolute 
alcohol, in which it is advisable to dissolve a pinch of 
methylene blue, and are then placed in methylene 
blue aniline oil (Solution 4) ; owing to the alcohol 
they will spread out well in the oil, and dehydration 
will be complete in a few minutes. If the sections have 
become very pale by this stage of the proceedings, 
rather more of the aniline methylene blue should be added 
than is directed above. After dehydration they are rinsed 
in pure aniline oil, which must then be extracted by 
thoroughly washing the specimens in xylol ; it is very im- 



lOO MEDICAL MICROSCOPY. 

portant that this be effectually done, otherwise the prepara- 
tions soon become brown and almost worthless ; two lots 
of xylol should be employed, the sections being transferred 
from one to the other. Finally they are placed on glass 
slides, and after draining off the superfluous fluid are 
mounted in balsam. The specimens may be counter- 
stained by immersing them in the safranin aniline oil 
(Solution 5) prepared in the same way as the aniline solu- 
tion of methylene blue; the time required varies from two 
to ten minutes, according to the dilution of the stain and 
the material to be colored. When removed from the 
stain they are immersed for a few seconds in pure aniline 
oil and then thoroughly washed in xylol. 

Although Klihne states that all bacilli, with the excep- 
tions named above, are colored by this method, the 
author has attempted in vain to stain the bacilli of pseudo- 
tuberculosis. 

*' Mast-zellen." — This seems to be the most suitable 
place to refer to these bodies. In sections of organs 
affected with diphtheria, glanders, and other processes, 
which have been stained by methylene blue or other 
aniline dyes, small, round, or spindle-shaped bodies are 
often met with ; they are about twice the size of lymph 
cells, and consist of coarsely granular protoplasm with 
a well-marked nucleus. The granules stain with the 
aniline dyes, but the nucleus remains uncolored, appear- 
ing as a bright spot in the midst of the deeply-stained 
protoplasm. These bodies occur in large numbers in the 
connective tissue, especially in the mucous membrane, 
and are generally situated in the neighborhood of the 
vessels. Their physiological and pathological significa- 
tions are not known. They are very numerous around 
rapidly growing tumors and also in elephantiasis. 

*' Mast-zellen " have hitherto been chiefly described by 



SELECTIVE STAINS. lOI 

foreign observers, and in England they are generally 
known under the term ^^ plasma cells." They are very 
liable to be mistaken for colonies of micrococci by those 
unfamiliar with them, but may be distinguished from the 
latter by the presence of the nucleus and by the irregu- 
larity in size of the component granules. 

Gram's Method of Staining." — This process is the 
one most generally adopted for staining micro-organisms. 
It will stain all the most important pathogenic bacteria, 
with the exception of those associated with cholera, 
typhoid fever, and glanders. 

The following solutions are required : — 

Soliitioji I. — Methyl aniline violet. This should always 
be freshly prepared. A few drops of pure aniline oil are 
placed in a medium-sized test-tube, which is then nearly 
filled with w^ater and thoroughly shaken, the thumb being 
placed over the mouth of the tube. The emulsion is 
filtered through a double-folded paper, and poured through 
a second time if the filtrate be not quite clear. A little of 
this aniline water is placed in a capsule, and to it is added, 
drop by drop, a concentrated alcoholic solution of methyl 
violet, until the mixture just becomes opaque. This stage 
is most easily recognized by placing the capsule on the 
edge of a piece of filter paper, and adding the dye until 
the margin is almost, but not quite, obscured. 

Solution 2. — Gram's solution. 

Iodine, ' I part, 

Iodide of potassium 2 parts, 

Water, 300 '' 

The sections are transferred from dilute spirit to methyl- 
aniline voilet, where they are allowed to remain for ^n^ 
or ten minutes ; after staining they are transferred to 
Gram's solution until they become brown and opaque : 



I02 MEDICAL MICROSCOPY. 

one minute is usually long enough. They are next re- 
moved to 60 per cent, alcohol, and gently moved about by 
means of a needle until no more color can be removed. 
Professor Crookshank's procedure may also be adopted, 
which is to transfer the specimens two or three times 
backward and forward between oil of cloves and the 
alcohol. When completely decolorized, the sections may 
be counter- stained by placing them in absolute alcohol to 
which two or three drops of a saturated alcoholic solution 
of eosin have been added ; they are at the same time 
dehydrated, and after clearing in cedar oil may be 
mounted in balsam. 

One caution is here necessary : if the specimens have 
been embedded in celloidin previous to cutting, oil of 
cloves must not be used for decolorizing, and it is better to 
dehydrate in 95 per cent, rather than in absolute alcohol. 

Weigert's Modification of Gram's Method. — 
Weigert and Kiihne were the first to observe that 
after staining bacteria in methyl violet and treating 
with Gram's solution and decolorizing with alcohol, 
much of the color was removed from the micro-organisms 
as well as from the tissues. These observers then proposed 
to use aniline oil for decolorizing, and Weigert conse- 
quently introduced the following modification of the 
method just described. 

The sections are first stained in lithium carmine as de- 
scribed on page 81, decolorized wiih acidulated alcohol 
and dehydrated with absolute alcohol. The sections are 
then transferred one by one to glass slides, and each is 
treated in the following way : — 

A couple of drops of methyl aniline violet freshly pre- 
pared (see above) are placed on the section and allowed to 
remain for about five minutes ; the excess is poured off, 
and a small quantity of Gram's solution poured on; after 



SELECTIVE STAINS. I03 

the lapse of one minute this is likewise poured off, and all 
moisture removed by gently pressing a folded filter paper 
on the specimen. Pure aniline oil is now added, and 
made to flow gently to and fro by tilting the slide ; the 
color will be removed at first rapidly and then more 
slowly, and fresh portions of oil must be used until no 
more color comes away. It occasionally happens that the 
section adheres to the glass all round, but not in the centre, 
so that some of the stain may be thus retained ; if such be 
the case, a corner of the section must be gently raised with 
a needle so as to permit some of the oil to flow beneath it, 
care being taken to prevent the whole section from being 
floated away. 

Decolorization being complete, two or three lots of 
xylol are poured over the section, a light cloud will be ob- 
served at first, and the xylol must be used until this has 
quite disappeared; unless this part of the process be thor- 
oughly carried out, the section will speedily turn yellow. 
A few seconds are allowed to elapse for the xylol to evapo- 
rate, and finally a drop of balsam is placed on the speci- 
men and a cover-glass applied. 

The lithium carmine solution is very apt to develop 
micro-organisms, and errors often arise on this account, 
the observer imagining that he has an extremely fine pre- 
paration. Some sections, such as of cirrhosis of the liver, 
known to be free from germs, should therefore be passed 
through the above process so as to ascertain if the carmine 
solution be untainted. 

The above process is particularly suitable for staining 
anthrax bacilli and sections of ulcerative endocarditis. In 
the latter not only are the masses of micrococci beautifully 
demonstrated, but a delicate purple tint is left in the layers 
of fibrin. 

Neelsen-Ziehl Method for Staining Tubercle 



I04 MEDICAL MICROSCOPY. 

Bacilli. — In order that this process may be carried out as 
successfully as possible, the specimens should be hardened 
in alcohol, or, if any of the chrome solutions have been 
used, the hardening material must be thoroughly extracted 
by water and the specimens soaked in alcohol for two or 
three days. Sections which haye been cut in celloidin or 
paraffin will also be found more satisfactory to manipulate 
than those which have been frozen, as these last are very 
apt to curl up in the acid, and it is difficult to spread them 
out again. 

As stated in the chapter on the ^^Examination of Spu- 
tum,'* this method depends on the property which the 
tubercle bacilli possess of resisting for some time the action 
of dilute acids, which distinguishes them from all other 
bacilli, except those of leprosy, which act in the same 
manner; but as in this country leprosy is fortunately a 
very rare disease, this method of staining may be said to 
be an absolutely distinctive one. Tubercle bacilli also stain 
by Gram's and Kiihne's methods, but these are never used 
for the purpose. I have used the double name for this 
process, for although introduced independently by the 
two observers, the processes are essentially the same. 

The solutions required are : — 

Solution I. — Carbol. fuchsine (Neelsen-Ziehl solution). 

Fuchsine, i part, 

dissolved in a 5 per cent, watery solution of 

Carbolic acid, loo part?, 

Alcohol, 10 " 

Solution 2. — Sulphuric acid, 25 per cent, watery solu- 
tion. 

Solution 3. — Methylene blue : — 

Methylene blue, 2 parts. 

Alcohol, 15 " 

Water, 85 '< 



SELECTIVE STAINS. I 05 

The sections having been soaked in dilute alcohol, are 
placed in the fuchsine solution contained in a covered 
capsule, where they should remain for at least an hour, or 
preferably for twelve hours. They are then decolorized in 
the sulphuric acid. This process is carried out thus : — 
Sections are allowed to remain in the acid for about 
thirty seconds, and are next washed in a large capsuleful 
of 60 per cent, alcohol, being gently moved about with a 
glass rod. Part of the color will be probably restored. 
If so, they must be returned to the acid for about ten to 
fifteen seconds, then again washed in the alcohol, this pro- 
cess being repeated until only a faint lilac tint remains. 
They are next counter-stained in the methylene blue solu- 
tion. An immersion of half a minute will be sufficient, 
after which they must be again washed in water. The 
blue stain is rapidly removed by the water, and in about 
half a minute the sections should be transferred to absolute 
alcohol. Still more color will then be abstracted, and the 
specimens must therefore be watched, or they will become 
completely decolorized. As soon as they assume a light 
blue tint they should be removed to cedar oil for clearing 
and finally mounted in balsam. The bacilli will be found 
to be stained a brilliant red, whilst the nuclei of the tissues 
are colored blue. This stain is very permanent, lasting for 
years. 

A more simple, but not so reliable a method consists in 
immersing the sections for several hours in Gibbes' 
double stain, and subsequently washing out the excess 
of color in methylated spirit, dehydrating in absolute 
alcohol, clearing in cedar oil, and mounting in balsam. 
Gibbes' double stain may be procured at any of the micro- 
scope makers. Its composition is rather complicated : — 

Rosaniline hydrochlorate, 2 parts, 

Methylene blue, i part. 

10 



Io6 MEDICAL MICROSCOPY. 

The mixture must be triturated, and three parts of 
aniline oil mixed with 15 parts of rectified spirit slowly- 
added, followed by 15 parts of distilled water. 

Methods of Staining Actinomyces. — This fungus 
has been more frequently met with during the last few 
years than it was formerly, not because cases have been 
any more numerous, but on account of discharges from 
the chest and other parts being more carefully examined in 
doubtful cases. 

For demonstrating the fungus in sections I have found 
the following method most satisfactory. The sections are 
first stained in rubin (see p. 87) for ten minutes, are then 
washed in water and placed in absolute alcohol for about 
three minutes. If the sections are seen to become very 
pale they must be taken out sooner. They are next trans- 
ferred, one by one, to glass slides, and stained according to 
Weigert's modification of the Gram method (see p. 102) 
On examination the clubs of the actinomyces will be found 
to be stained red, whilst the mycelium appears violet. 

Plaut's Method, which is the one usually adopted, is 
as follows : The sections are placed for two hours in 
Neelsen's solution (see p. 104). The time of staining may 
be shortened to twenty minutes if the fluid is maintained 
at a temperature of 40° C. in an incubator. The sections 
are next washed in water and immersed for ten minutes in 
a saturated alcoholic solution of picric acid. After washing 
for twenty minutes in water they are transferred for about 
fifteen minutes to 50 per cent, alcohol. In about a quarter 
of an hour they will be properly decolorized, and may 
then be dehydrated in absolute alcohol, cleared in cedar 
oil and mounted in balsam. By this method the clubs 
only are stained, the mycelium remaining uncolored. 

I now come to consider a small class of selective stains 



SELECTIVE STAINS. I07 

which are dependent upon micro-chenaical reactions. 
These are practically confined to two, namely, the methods 
for demonstrating waxy degeneration (amyloid reaction), 
and for the exhibition of iron in the tissues. 

The Amyloid Reaction. — There are several meth- 
ods for showing this reaction, by means of which portions 
of tissue which have undergone waxy degeneration are 
clearly distinguished by change of color from the normal 
portions. The chief processes are as follows : — 

1. A thin section of the tissue is placed on a slide, and 
a drop of an aqueous solution of iodine upon it. The 
specimen may then be mounted in the iodine-mounting 
fluid (see p. 114). The edge of the cover-glass being fixed 
by a ring of Dammar varnish. On examination the 
affected portions appear as dark yellow or brown, the 
remainder of the section being light yellow. 

2. This was Virchow's original method. The sections 
as before are dipped into a watery solution of iodine, and 
are then conveyed to a 2 per cent, solution of sulphuric 
acid. As soon as the blue colorization appears, the sec- 
tions are swilled in water, and mounted in Farrant's solu- 
tion. 

3. Methyl Aniline Violet. — The stain is made as described 
on p. loi. The sections are placed in it for about five 
minutes, are then washed in water for twenty-four hours, 
and mounted in Farrant's solution. The amyloid sub- 
stance will be found to be colored red, the rest of the 
tissue being a dull blue. The preparations will not keep 
well in the mounting medium unless they have been pre- 
viously fixed for half an hour in a one per cent, solution 
of perchloride of mercury. 

4. Iodine Green. — The sections are soaked in this stain 
(see p. '^6) for ten minutes, then washed in water and 
mounted in Farrant's solution. Very beautiful results are 



Io8 MEDICAL MICROSCOPY. 

thus obtained, the affected portions being colored a rose- 
pink, whilst the remainder assume a bluish-green color. 

Iron Reaction. — Iron is occasionally found free in 
the liver and other organs in diseases in which excessive 
destruction of red blood corpuscles is brought about. Dr. 
Mott, in 1889, before the Pathological Society, showed 
some beautiful sections of liver from a case of pernicious 
anaemia. The iron was seen in the form of blue granules 
(converted by ferrocyanide of potassium into Prussian 
blue) in the cells and capillaries of the portal zone. 

The method for obtaining such specimens is as fol- 
lows : The organs must be hardened in alcohol, and after 
sections have been cut, they are placed in a solution of 
ferrocyanide of potassium acidulated with hydrochloric 
acid. In a very few minutes the sections will turn blue, 
and may then be removed and mounted in glycerine. An 
objection to this method lies in the partial solubility of the 
Prussian blue, which escapes and tinges other parts of the 
tissue a similar color. 

More exact definition of the deposit may be obtained 
by exposing the sections to a watery solution of sulphide 
of ammonium, which precipitates the iron as dark greenish 
granules, or by placing them in a solution of sulphocyanic 
acid, when the granules will be of a blood-red color. 

Before concluding the chapter, this seems to be the most 
suitable opportunity for referring briefly to the principal 
method which is employed for demonstrating the karyo- 
kinetic figures in nuclei ; it is known as Flemming's 
method. 

The organ to be investigated must be removed from the 
body immediately after death ; the larynx, after an opera- 
tion for excision, for instance, is very suitable for this pur- 
pose. The tissue must then be '* fixed," so that the vari- 
ous components are made to retain their living structural 



SELECTIVE STAINS. IO9 

appearance. Flemming's fixing mixture has the following 
formula : — 

Chromic acid (l per cent, solution'! .... 15 parts, 
Osmic acid (2 *' " " ) . . . 4 " 
Acetic acid i part. 

Small pieces of the tissue are placed in this solution for 
two days, and are then washed in a stream of running 
water for at least an hour. They are afterwards hardened 
in alcohol, for which purpose three days are required. 
They may then be embedded in celloidin or paraffin (the 
former being preferable) and sections cut. The best stain- 
ing fluid is undoubtedly safranin (see p. 86), after which 
they are washed in acidulated alcohol (see p. 82), then de- 
hydrated in absolute alcohol, cleared in oil of cloves and 
mounted in balsam. 

Most of the nuclei will by this method be almost de- 
colorized, whilst the karyokinetic figures will be stained a 
brilliant red. 

Another process for demonstrating these figures which 
produces very pretty results, but not quite so well defined 
as with Flemming's method, is, after hardening the tissues 
in alcohol to stain them by Gram's method (see p. loi), 
when those nuclei which are undergoing division will be 
colored a deep purple, whilst the others will be almost 
decolorized. 



CHAPTER VII. 
CLEARING AND MOUNTING. 

The final stage in the preparation of microscopic speci- 
mens is perhaps the most difficult, and requires more 
practice than any of those previously described. It 
not unfrequently happens that after a section has been 
successfully cut and stained, it is irretrievably damaged 
in spreading it out on the slide. Skill in manipulation 
can only be obtained after much practice, and each 
worker will probably arrange methods of his own, but 
a few general hints may be of service in indicating the 
more common errors and modes of correcting them. 

In order that a section may be satisfactorily examined 
its interstices must be filled by some material having a 
refractive index higher than that of air, in order to 
prevent the irregular reflection resulting from the want 
of homogeneity. Before these reagents can permeate the 
tissues, the sections have to be cleared. The liquids 
employed for this purpose have also a high index of re- 
fraction, by which the tissues are rendered transparent, 
and at the same time the alcohol which has been used for 
dehydration is removed, and the balsam or other resinous 
media can easily flow in. 

Two varieties of mounting media are employed, namely, 
fluid and solid, and the methods for employing these differ 
very much from one another. We will consider first the 
use of fluid media. 

After the completion of the staining process the 

no 



CLEARING AND MOUNTING. Ill 

sections are placed in water ; each one is then removed 
to a glass slide by means of the section lifter. The 
specimen must be carefully spread out on the lifter by 
means of needles, and then very cautiously pulled up out 
of the water. Unless this is slowly done, the section 
is nearly sure to curl up, or float off from the lifter. 
Having successfully removed the latter, with the former 
adhering to it, the lifter is laid on the centre of the glass 
slide, and one corner of the section having been fixed by 
means of a needle, the lifter is cautiously pulled from 
under it and not the section from off the lifter. 

Sometimes in spite of all care, especially if the section 
had been cut by freezing, the specimen will obstinately re- 
fuse to lie properly on the lifter, whilst this instrument is 
being removed from the water. A very good expedient 
is to place a clean slide in the dish containing the water, 
and then to spread a section on it by means of a needle, by 
gently steadying it thus, the slide may be raised, and so 
the specimen lifted out lying perfectly flat. 

The slide is now tilted, so as to allow superfluous fluid 
to strain off, and as much water as possible is removed 
by placing a piece of folded filter paper near the section, 
or if the object be not too delicate, the paper may be 
gently pressed on it. A drop of the mounting fluid is 
then added, and a cover glass laid on it in the following 
manner. The cover glass is taken between the finger and 
thumb of the left hand and placed with one edge down- 
ward, just beyond the drop of fluid. The upper edge of 
the glass is supported by a needle which is gradually 
lowered until the glass comes in contact with the liquid. 
If this be carefully done, no air bubbles will be included, 
but if this should happen the glass must be lifted up, and 
a fresh one applied. The amount of fluid should be suffi- 



112 MEDICAL MICROSCOPY. 

cient so as just to occupy the space beneath the cover- 
glass, as any excess interferes with the subsequent applica- 
tion of the fixing material. This quantity can only be 
gauged by practice. 

The cover-glass having been placed in position, it 
must be fixed by one of the cementing materials presently 
to be mentioned. For this purpose the glass slide should 
be held in the left hand. Any excess of mounting fluid 
which has escaped must be removed with a soft cloth. 
A glass rod is then dipped in the cementing fluid and 
gently passed round the edge of the cover-glass. This 
should be done by holding the rod a fraction of an inch 
above the glass so as to avoid touching it, but at the same 
time be near enough to guide the fluid. The rod will pro- 
bably have to be dipped in the liquid two or three times 
before the circuit of the glass is complete. It is needless 
to add that only a very narrow portion of the cover-glass 
must be included in the ring; but the same precaution 
need not be taken with the glass slide. As the cement 
hardens a second or third coating should be applied, so as 
to form a raised border. 

The most common fluid media employed must now be 
considered. 

Glycerine, — This is an excellent medium for sections 
which have been previously hardened and are stained in 
dyes which are easily extracted by means of alcohol. It 
is also suited for specimens such as those containing fat 
which would be rendered useless by dehydration, and 
further, for those sections which have been treated with 
metallic salts. 

It has the great disadvantage of causing fibrous tissue to 
swell up, and so lose its characteristic structure. In mount- 
ing fresh specimens this difficulty may be overcome by 



CLEARING AND MOUNTING. II3 

covering them the first day with equal parts of glycerine 
and water, and on the following day removing the cover- 
glass and adding pure glycerine. 

This reagent is much used for preserving parasites. For 
this purpose such specimens should be immersed in equal 
parts of glycerine and water, contained in a small covered 
capsule for twenty-four hours. On the second day a small 
portion of the liquid must be removed with a pipette, and 
a few drops of glycerine added. This process being 
repeated day by day until the parasite is surrounded by 
the pure reagent. 

Another excellent way of treating delicate objects is to 
employ a mixture of glycerine, alcohol and water. By 
the evaporation of the alcohol the liquid gradually in- 
creases in density, and after some time a cover-glass may 
be applied and fixed, or the object brought into pure 
glycerine. 

Lee recommends the following mixture : — 

Glycerine, I part, 

Alcohol, . . . . I " 

Water, 2 parts. 

Glycerine Jelly. — This should be procured already 
prepared. The bottle containing the reagent is placed in 
hot water until the contents are fluid. The object having 
been placed on the slide and dried, the latter is slightly 
warmed and a drop of the jelly added. The cover-glass 
is then applied and gently pressed down, any air bubbles 
being expelled by pressure with a glass rod. 

The jelly soon solidifies and fixes the glass, but it is 
more secure to protect the preparation with a ring of 
cement. This reagent is useful for vegetable substances 
and for parasites. 



114 MEDICAL MICROSCOPY. 

Glycerine Mounting Fluid. — 

Camphor water, 2 parts, 

Glycerine, I part, 

Pure gum arable, Iij4 parts. 

This preparation is chiefly used for sections which have 
been injected with Prussian blue. The method of using it 
does not differ from those of the other fluid media. 

Farrant's Solution. — Farrant's solution being very 
difficult to prepare is best procured ready made. It is an 
extremely useful reagent combining most of the advantages 
of glycerine, with but few of its disadvantages. Sections 
preserved in it, however, are apt to become cloudy after 
some years. 

Sections are mounted in the usual way, and after some 
days the fluid dries at the edge of the cover-glass, so fixing 
it; but it is better to add a ring of Hollis's glue, or zinc- 
white cement. 

It is especially useful for mounting fresh specimens or 
sections which have been stained to demonstrate the amy- 
loid reaction (see p. 107). 

Iodine Mounting Fluid. — 

Glycerine, i^^ . 6 parts. 

Liquid iodi (B. P.), 31^" 

Water, 6 " 

The ingredients are mixed and six parts of gum arable 
added. 

This preparation is only employed for sections stained 
in iodine. The method of using it is the same as for 
glycerine. 

Two other fluid media may be mentioned, namely, 
acetate of potassium in a saturated solution for 
mounting vegetable tissues or those exhibiting fatty 



CLEARING AND MOUNTING. II5 

change; and castor oil for mounting crystals which are 
soluble in Canada balsam. 

CEMENTING MATERIALS. 

A section having been mounted in one of the fluids just 
mentioned, a ring of cement is applied to fix the cover- 
glass. If a square one has been used the cement is applied 
in the manner already described (see p. 112) ; but if the 
glasses are circular a Shadboldt's turn-table may be 
employed. 

The most usual materials employed are Canada balsam, 
which must be liquid enough to run easily, or Dammar 
varnish (see p. 120). 

Other cementing substances are : Brunswick black, 
Hollis's marine glue and gold size. Any of the above 
may be supplemented by a ring of zinc white. 

All these materials are best secured ready prepared, as 
their composition is complicated and not readily carried 
out. 

For sections mounted in glycerine, Dr. Woodhead 
(^* Pathological Histology,'* p. 80) strongly recommends 
the method suggested by Dr. Marsh, namely, a solution of 
gelatine; the rationale of the process being that gelatine 
readily mixes with the glycerine in its imme Hate neigh- 
borhood. The solution is prepared by placing a small 
quantity of gelatine in a narrow glass beaker, covering it 
with water, and allowing the gelatine to take up as much 
of the water as it will. Any superfluous water is poured off 
and the mixture is heated and three or four drops of creo- 
sote are added to each ounce of the fluid. The mixture 
will then solidify, and each time it is needed, the bottle 
containing it is immersed in a cup of warm water to render 
the contents fluid. All superfluous glycerine must be 
removed by the aid of a camel-hair brush and a damp 



Il6 MEDICAL MICROSCOPY. 

cloth. A ring of the gelatine fluid is painted round the 
edge of the cover glass ; as soon as this is set it is painted 
over with a solution of bichromate of potash, made by 
dissolving ten grains of that salt in one ounce of distilled 
water. Dr. Marsh recommends that '* this application of 
bichromate of potash, should be made in the daytime, as 
the action of daylight upon it, in conjunction with the 
gelatine, is to render the latter insoluble in water.'* 

The preparation is finished by washing it over with 
methylated spirit to remove all the glycerine, and then a 
ring of zinc-white is applied. 

Mounting in Solidifying Media. — This is accom- 
plished by preserving the sections in a transparent refract- 
ing material which solidifies on drying into a vitreous 
mass. Various resins are employed, dissolved in a fluid 
which easily evaporates. 

As the substances are insoluble in water this must be re- 
moved. This process is known as '^ dehydration.'' 

After staining and washing, the sections are placed in 
absolute alcohol, by which means all the water is rapidly 
extracted. The time usually required is five minutes, 
but if the specimens are thick a longer period may be 
necessary. 

Great care is needed in the dehydration of sections em- 
bedded in celloidin. As the material is soluble in abso- 
lute alcohol it is advisable to employ 95 per cent, alcohol 
to abstract the water, allowing about ten minutes for the 
process. 

Very delicate specimens may be treated thus : A clean 
cover-glass is placed at the bottom of a shallow dish con- 
taining water, and the sections floated on to it. By means 
of needles one specimen is spread out on the cover-glass, 
which is removed with a pair of forceps, and two or three 
lots of absolute alcohol poured over it. The spirit is 



CLEARING AND MOUNTING. II7 

allowed to evaporate, and a clearing solution applied in 
the same way. Finally the section is mounted in balsam. 

In the ordinary course of events each specimen is re- 
moved from the alcohol on a section lifter and immersed 
in one of the clearing agents. Owing to the rapid escape 
of the alcohol the section flattens out and floats on the 
surface of the fluid, but it should be gently forced below 
the surface with a needle. The specimen will soon be- 
come perfectly transparent. If any cloud appears, or if 
white spots are seen (this being best tested by viewing it 
against a black surface, such as the sleeve of a coat), de- 
hydration has been inefficiently performed, and the section 
must be again placed in the absolute alcohol. 

When perfectly transparent each section is removed to a 
clean glass slide by means of a lifter which is laid flat on 
the slide. One corner of the specimen is then fixed with 
a needle, and the lifter cautiously withdrawn. If this has 
been done with skill the section will be almost flat. If 
any part of it has become rolled up, or wrinkled, this de- 
fect must be remedied by means of two clean needles, the 
one being used to fix the section, and the other to remove 
the creases. This is rendered easier by having an excess 
of the clearing reagent on the slide. 

The slide is now tilted to allow the clearing fluid to run 
off. This must be done gently, and if necessary the sec- 
tion steadied by a needle to prevent its slipping off. 
After draining, the superfluous oil is removed with a cloth, 
or a folded filter paper may be laid on the section and 
gently pressed. 

A drop of Canada balsam or Dammar varnish is then 
placed on the section and a clean cover-glass applied in 
the same manner as described on p. iii. By this means 
the intrusion of air bubbles is prevented as far as possible. 



Il8 MEDICAL MICROSCOPY. 

but should any occur an attempt may be made to remove 
them by gentle pressure on the cover-glass. Very often 
this is unavailing, and it is better to leave them alone, as 
they are generally expelled by the contraction of the 
balsam, and undue pressure may either damage the section, 
or break the cover-glass. 

Occasionally after two or three days gaps are noticed 
beneath the cover-glass which may even trespass upon the 
section. The balsam must then be liquefied by gently 
warming the slide, and a drop of balsam placed on the 
edge of the cover-glass nearest the gap. A little pressure 
will drive out the air, the resin taking its place. 

For some days after mounting, the slides should be 
allowed to lie flat, protected from dust, and when the 
balsam has thoroughly set, they may be stored edgeways. 
Slides should be labeled as soon as finished, the descrip- 
tion including the name of the organ, or nature of the 
tissue, the disease, method of staining and date. 

We now have to consider the various clearing reagents 
in use. 

Any of the essential oils will answer the purpose, but 
those most commonly employed are, oil of cloves, creo- 
sote, cedar wood oil, xylol, and oil of bergamot. 

Oil of Cloves. — Although more generally employed 
than any of the others, it has several disadvantages; it 
certainly produces excellent results, but has a clinging 
and penetrating odor, rapidly dissolves celloidin, and 
removes the aniline dyes, picric acid, and eosin. If 
sections be exposed too long to its action, they become 
rigid and brittle, and should therefore be removed directly 
they are transparent. 

Creosote. — I prefer this to any of the others for 
ordinary microscopic work, except for use with specimens 



CLEARING AND MOUNTING. II9 

stained in the aniline dyes. Its odor is not disagreeable, 
sections may remain in it for any length of time without 
becoming brittle, and it does not dissolve celloidin. 

Cedar Wood Oil. — For bacteriological purposes this 
is undoubtedly the best clearing reagent. It does not 
dissolve celloidin, or abstract the aniline dyes. It has, 
however, rather a tendency to stiffen the sections, espe- 
cially if they are left in it for some time. 

Xylol. — This is not very often used, except in special 
methods, such as Weigert's modification of the Gram 
method, and Kiihne's processes for the demonstration 
of bacteria in the tissues. 

Oil of Bergamot. — This is a good clearing agent, 
and has the advantage of not dissolving celloidin or 
affecting the aniline dyes. It has, however, a very dis- 
agreeable smell. 

Turpentine, Oil of Cajeput, and Oil of Origanum, 
are sometimes employed, but possess no advantage over 
the others. 

Practically the only two resins employed for mounting 
specimens are Canada balsam and Dammar varnish. 

Canada Balsam. — This is suitable for all specimens 
which will bear dehydration in alcohol, and clearing with 
an essential oil. It may be procured dissolved in various 
fluids. The most common being turpentine or chlo- 
roform, or a mixture of the two. Benzol also is sometimes 
employed. 

Any of these solutions are suitable for ordinary sections, 
but for bacteriological work, or for sections stained in the 
aniline dyes, xylol is decidedly the best solvent. Balsam 
dissolved in xylol may be procured from most of the firms 
that supply microscopical apparatus, or any of the ordinary 
solutions may be evaporated to dryness over a water bath, 



120 MEDICAL MICROSCOPY. 

taking care that the balsam does not get over-heated and 
become brown. The dried resin is dissolved in xylol. 
The consistency of the solution should be such that 
it flows easily, that is to say, it should be nearly as thin as 
glycerine. 

Taking everything into consideration this is the best 
kind of balsam to use. It should be kept in a special 
^^ balsam bottle," which consists of a wide-mouthed 
bottle furnished with a ground glass cap and a small 
glass rod lying in it. 

Dammar Varnish. — It is also well to procure this 
ready made, but its composition is as follows: — 

Gum dammar, 2 parts, 

Gum mastic, i part, 

Turpentine, 4 parts, 

Chloroform, .... 2 '* 

The gums are mixed with the solvents until they are 
dissolved, and the mixture filtered through cotton -wool. 
This preparation has the advantage of being quite 
colorless, and is therefore useful for photo-micrography. 
Sections preserved in it do not keep well, becoming 
after a time cloudy and granular. It must not be used 
for sections stained in the aniline dyes, or for those 
containing micro-organisms. 

Breaking Down Old Specimens. — In the course 
of preparation of a number of specimens a certain pro- 
portion will from one reason or another be spoiled. The 
glass slides and cover-glass need not be wasted, but a 
jar should be kept into which waste alcohol should be 
thrown, such as that used for dehydration. Into this 
rejected slides may be put from time to time, and after 
soaking for a week or two may be removed, and will 
then be quite suitable for another occasion. 



CLEARING AND MOUNTING. 121 

At Other times valuable specimens may be damaged 
in some way; the cover-glass may be broken, or the 
sections may have faded. A section which has been 
mounted in glycerine is easily released by cutting 
through the cementing material with a knife, and then 
placing the slide in water, when the section will float 
off. It may then be stained, and mounted again in the 
usual way. 

One which has been preserved in balsam must be 
immersed for some time in chloroform. After some 
hours the section will be freed, and should then be 
allowed to lie in absolute alcohol for a day. It may 
subsequently be restained and remounted. 



II 



CHAPTER VIII. 

COMPLETE PROCESSES FOR THE PREPARATION OF 
SECTIONS. 

In this chapter it is proposed to give as briefly as possi- 
ble two methods of preparing sections. In the preceding 
chapters all the processes have been considered in detail, 
and should be carefully studied before this chapter is con- 
sulted. Whilst working I have found it convenient to 
have a summary of the various stages through which the 
specimens have to pass before they are complete, and give 
two of them here ; others may be compiled by referring to 
what has already been described. 

Method I. — Harden in Miiller's fluid, cut by freezing, 
stain in hsematoxlyin and eosin, mount in Canada balsam. 

1. Place the specimen, removed as soon after death as 
possible, in a bottle containing several times its bulk of 
Miiller's fluid. Change in twenty-four hours, merely 
pouring the fluid off without removing the specimen from 
the bottle. Change again on the third and seventh days, 
and at the end of each week until the sixth. Examine to 
see if the specimen is sufficiently hardened, if this be the 
case, transfer to dilute spirit (fifty per cent.) until wanted, 
if not, put back in Miiller's for another week. 

2. Place the specimen in water until it sinks, and the 
washings are no longer colored. Transfer to solution of 
dextrin (see p. 56) for twenty-four hours. Remove the 
specimen, by means of forceps, to the plate of an ether 
freezing microtome. Work the spray cautiously so as not 



PREPARATION OF SECTIONS. 1 23 

to overharden. Float the specimens off the knife into 
water. 

3. Take a dihite solution of hsematoxylin and allow 
sections to remain in it for several hours, or in a strong 
solution for about three minutes (p. 79). Wash the sec- 
tions in water and allow them to float exposed to the light 
for two or three hours. 

4. Transfer to absolute alcohol, containing a few drops 
of alcoholic solution of eosin, and allow the section to 
remain for five minutes ; or soak it in a watery solution of 
eosin for ten minutes and then place in absolute alcohol 
for three minutes. 

5. Clear in creosote. 

6. Mount in Canada balsam. 

Method II. — Harden in alcohol, embed in celloidin, 
stain in carmine and picric acid, mount in Canada balsam. 

1. Cut small portions of tissue and place them in abso- 
lute alcohol. Change this solution in twenty-four hours. 
The specimens will be hardened in from two to four days, 
depending upon their size, and may be left in the alcohol 
for an indefinite period. 

2. Place the specimens in equal parts of absolute alco- 
hol and ether for twelve hours ; transfer to celloidin of the 
consistence of mucilage for twelve hours; place each 
specimen on a dry cork, and allow celloidin to set ; then 
immerse in a large jar containing eighty per cent, alcohol, 
for at least twelve hours. Select one specimen and place 
in the clamp of the microtome. Be careful to have the 
knife and specimen constantly covered with an excess of 
dilute alcohol (fifty per cent.). Transfer sections, when cut, 
by means of a cameFs-hair brush, to water, where they will 
spread out, then keep them in dilute spirit. 

3. Place the sections in lithium carmine (p. 81) for ten 
minutes. Wash in acidulated alcohol (p. 82) until the 



124 MEDICAL MICROSCOPY. 

sections are a light pink, or until no more color comes 
out. 

4. Transfer the sections to absolute alcohol containing 
a few drops of a saturated alcoholic solution of picrid acid 
for five minutes. 

5. Clear in cedar oil. If after the preparation of one 
specimen the contrast stain is found not to be strong 
enough, more picric acid must be added to the alcohol. 
If white patches are noticed in the sections, dehydration 
has not been properly accomplished, and the sections 
must be put back into the absolute alcohol. 

6. Mount in Canada balsam. 



CHAPTER IX. 

METHODS OF PREPARING SECTIONS OF THE CENTRAL 
NERVOUS SYSTEM. 

In order to prepare sections of the brain or spinal cord, 
very complicated and rather difficult methods have to be 
adopted. It has, therefore, been thought advisable to de- 
vote a separate chapter to their consideration, and to 
describe in detail the various processes from the time that 
the cord or brain are removed from the body, until the 
sections are complete. As the examination of the spinal 
cord is more frequently required than that of the brain, 
the following description will be understood to apply to 
the cord unless otherwise stated : — 

Hardening. — The spinal cord should be removed from 
the body as soon after death as possible, and there will be 
less chance of injury if it be removed from the front 
instead of from the back, as is usual. The method of thus 
doing not being generally known, a brief outline of the pro- 
cedure is here given. It is as follows : The viscera having 
all been removed, the inter-vertebral substance above and 
below the second lumbar vertebra is cut through, the 
pedicles of the vertebra divided with bone forceps, and 
the body then wrenched out with lion forceps. A specially 
devised chisel having a point projecting from its cutting 
edge is inserted with this point in the vertebral canal, and is 
driven with a mallet through the entire line of pedicles on 
the right side to the base of the skull ; a like manoeuvre is 
performed with a similar chisel on the left side. The 

I2S 



126 MEDICAL MICROSCOPY. 

bodies of the vertebrae having been removed, the theca is 
fully exposed and is slit up with probe-pointed scissors. 
The cord is removed by dividing the nerves on either side 
and those of the cauda equina, and gently lifting it, with 
the membranes out of the canal. 

Transverse cuts are made at intervals of half an inch 
if the whole cord is intended for preservation or the 
diseased portion is separated from the healthy. 

The best fluid for hardening purposes is a solution of 
bichromate of ammonium (two per cent.) or Muller's fluid. 
The former is more rapid in its action, but the latter 
more regular, and is therefore to be preferred. The 
cord should be suspended in a tall jar, which should be 
large enough to contain sufficient fluid to measure several 
times the bulk of the specimen. The hardening reagent 
must be changed at the end of twenty-four hours, on the 
third and the seventh days, and at the end of each week 
until the process is completed, the jar in the meanwhile, 
with its contents, being kept in a cool place and in the 
dark. Five or six weeks will probably be required, and 
the cord should be tested from time to time to see if it is 
properly hardened. To do this, a small section of the 
specimen should be taken, and placed in water; it ought 
to remain flat, if it curls up it is not yet ready. 

Exposure to Muller's fluid generally renders the tissue 
brown in color. When sufficiently hardened, the cord 
is washed in water and steeped in eight per cent, alcohol, 
where it may remain until arrangements are made for 
cutting. For the sake of convenience the specimen may 
be cut into segments, which can be placed in small bottles 
carefully labeled. 

Cutting. — Sections may be cut by freezing, but this 
will be found by no means satisfactory, and it is far 
better to embed the cord in celloidin or paraffin. The 



CENTRAL NERVOUS SYSTEM. 1 27 

choice of these two will depend upon many circum- 
stances, in a great measure upon the microtome it is 
intended to employ. If a series of sections are to be cut 
by the Cambridge Rocking microtome, paraffin is un- 
doubtedly to be preferred, but if any other microtome be 
selected celloidin should be chosen. 

The method of embedding in paraffin does not differ 
from that described on p. 60. For celloidin, after re- 
moval from the hardening reagent the specimens are 
placed in absolute alcohol for two days, and are then 
transferred to equal parts of ether and alcohol, for at 
least twelve hours. They are next immersed in a thin 
solution of celloidin for some hours, and afterwards in 
another solution about* the consistence of mucilage. 
After another twelve hours they are deposited upon 
pieces of cork a little larger than the specimens, and 
when the celloidin has commenced to set, the corks are 
thrown into a large jar containing eighty per cent, alcohol, 
care being taken that the celloidin is below the surface 
of the liquid. In twelve hours, sections may be cut by 
Reichert's or Schanze's microtome. 

Hamilton has introduced a method by which sections, 
which have been embedded in celloidin, may be cut on 
a freezing microtome. The specimens must be removed 
from the corks and placed for forty-eight hours in 
Erlicki's fluid, which has the following composition : — 

Potassium bichromate, 2.5 parts, 

Sulphate of copper, 5 part, 

Water, 100 parts. 

This is done in order to free the preparations from 
the spirit, and they are then placed in the following 
mixture; — 



128 MEDICAL MICROSCOPY. 

Sulphate of copper, .5 part, 

Potassium bichromate, 2.5 parts. 

Mucilage of syrup and gum, 100 " 

This solution, with the specimens, should be kept for two 
or three days in a warm chamber at t^S^ C. 

Sections are then cut in the freezing microtome, re- 
ceived in Erlicki's fluid, washed in methylated spirit, and 
stained according to Weigert's method presently to be 
described. 

If the specimens are cut with a Reichert's microtome, 
sections will, as a rule, be easily obtained, but if the 
attempts prove unsuccessful, the precautions and measures 
suggested on p. 69 should be carefully followed. 

For series-cutting the Cambridge Rocking microtome 
is undoubtedly the best, or series may be obtained by 
the process described on p. 70. It may so happen that 
after embedding in celloidin it is desired to cut series by 
the Rocking microtome. This may be done by removing 
the specimens from the corks, and immersing them in 
benzol, and then transferring to the liquid wax as 
described on p. 57. They may next be fixed to the 
Rocking microtome in the usual manner. 

Staining. — Nearly all specimens of the central nervous 
system are now stained by one of three methods, namely, 
those of Weigert or Pal, or in aniline blue black. 

For the demonstration of certain lesions the ordinary 
methods are preferable. For instance, when it is desired 
to examine miliary aneurisms in the brain, ammonium 
carmine should be used (see p. 84) or haematoxylin and 
eosin. 

A simple method which stains the ganglion cells very 
well, is to dilute Stephens' blue-black ink one half to two- 
thirds (Harris and Power). 



CENTRAL NERVOUS SYSTEM. 1 29 

As a rule, sections are made for the purpose of inves- 
tigating tracts of degeneration, and for this purpose one of 
the processes named above, and which we now proceed to 
describe, must be employed. 

Weigert's Method. — The solutions required are: — 

Solution I : — 

Saturated solution of acetate of copper, . . i part, 
Distilled water, I " 

Solution 2 : — 

Haematoxylin, I part, 

Alcohol (90 per cent.), 10 parts, 

Concentrated solution of lithium carbonate, . i part, 

Distilled water, 90 parts. 

The haematoxylin must be dissolved in the absolute 
alcohol, the water added, and the mixture boiled. After 
it is cool the lithium carbonate is added. This solution 
will not be ready for use for a couple of days, and will not 
keep for more than a month. 

Solution 3 : — 

Borax, 2 parts, 

Ferricyanide of potassium, 2.5 " 

Water, 200 " 

It is essential that the specimens should have been 
hardened in MuUer's fluid. 

The first step consists in placing the sections, whether 
single or in series, in the solution of acetate of copper 
for twenty-four hours, and then thoroughly washing them 
for about half that time in water, which should be 
frequently changed. This part of the process may be 
performed in mass before sections are cut, by placing 
the specimens after embedding in the copper solution for 
two or three days, or if a warm chamber at 2^^'^ C. can be 

12 



130 MEDICAL MICROSCOPY. 

employed, forty-eight hours will be sufficient, sections 
being then cut. When the washing is complete the 
sections are placed in the haematoxylin solution for one or 
two days, and then removed to water. They should now 
exhibit a blue-black color, and be perfectly opaque. If 
this be not the case, a drop or two of a saturated solution 
of lithium carbonate may be added to the water, and if the 
desired appearance is not then produced, the specimens 
must be returned to the hsematoxylin solution for another 
twenty-four hours. They must next be thoroughly washed 
in water, which should be changed every few hours, until 
the washings are no longer colored. Unless this process 
be thoroughly carried out the sections will ultimately 
develop a number of dark spots which will render them 
practically useless. 

The next step consists in differentiating them. For this 
purpose the sections are placed in the borax and potassium 
ferricyanide solution (Sol. 3). They will at once be seen 
to undergo a change, becoming lighter in color, and the 
gray matter gradually becoming separated from the white. 
This part of the process must be carefully watched, and as 
soon as the two parts of the cord are clearly defined they 
must be removed to water. The time required varies from 
five minutes to half an hour. If left too long the haema- 
toxylin will be removed and the specimens appear uni- 
formly brown. 

After washing in water the sections must be dehydrated, 
cleared in creosote, and mounted in balsam. If the speci- 
mens were previously embedded in celloidin, it will be 
better to use 95 per cent, alcohol for dehydrating, other- 
wise owing to the solution of the embedding material in 
the absolute alcohol, the sections will probably fly in 
pieces, and thus all the labor and trouble bestowed upon 
them will be lost. 



CENTRAL NERVOUS SYSTEM. I3I 

Stained in this manner, the white substance of Schwann 
assumes a purple-black color, whilst the connective tissues, 
ganglion cells, and axis cylinders are dyed an orange-brown. 
If there are tracts of degeneration (sclerosis) these also 
assume the latter color. 

Pal's Method. — This is a modification of the pre- 
ceding. 

The solutions required are: — 

Solution I : — 

Haematoxylin, .75 part, 

Distilled water, 90 parts, 

Alcohol, 10 " 

to which is added immediately before use a small quantity 
of a saturated solution of lithium carbonate in the pro- 
portion of three drops of the solution to ten c.c. of the 
haematoxylin stain. 
Solution 2 : — 

Permanganate of potash, . . . .25 per cent, solution. 

Solution 3 : — ^' Pal's solution." 

Oxalic acid, I part. 

Potassium sulphite, i " 

Distilled water, 200 parts. 

The specimens are hardened in Muller's fluid, as de- 
scribed above and embedded in celloidin, the sections 
being cut either singly or in series. They are at once 
transferred to the haematoxylin solution, where they re- 
main for five or six hours, and are then transferred to 
water containing a drop or two of a saturated solution of 
lithium carbonate. 

They should then be bluish-black in color and opaque. 

The sections must remain in water, which should be 
frequently changed, until no more color will come out. 

The method of differentiating is the characteristic part 



132 MEDICAL MICROSCOPY. 

of this process. The sections are first placed in the per- 
manganate of potash solution for from fifteen to twenty 
seconds; after which they are immersed in Pal's solution. 
Here they must be carefully watched until the white and 
gray matters are distinctly defined. This will probably be 
brought about in from one to two minutes. If any black 
spots appear the sections must be again placed in the per- 
manganate of potash for a few seconds, and then again 
washed in Pal's solution. 

After this they must be washed in water for a quarter 
of an hour. If they are now dehydrated (95 per cent, 
alcohol) cleared, and mounted in balsam, the medullary 
sheaths will be found stained a bluish-black color, the 
remainder of the tissue being white. 

Counter-staining is therefore advisable, and for this pur- 
pose borax carmine (see p. 85) is the best solution to use. 

Pal-Exner Method. — This process is more rapid 
than the preceding (although in some respects more trou- 
blesome), and produces very good results. 

The nervous material cut into small segments is placed 
whilst fresh in about ten times its bulk of one half per 
cent, solution of osmic acid, where it remains two or 
three days, the solution being changed daily. 

The specimens are then removed, washed in water, and 
dipped for a few seconds into absolute alcohol, and are 
then embedded in celloidin or paraffin. 

Sections are cut in glycerine and removed to water. 
They are differentiated in the same manner as in Pal's so- 
lution, that is to say in permanganate of potash, and in 
the liquid known as Pal's solution {vide supra). 

Aniline Blue-Black. — By staining sections of the 
central nervous system by this method, the nerve cells are 
especially brought into prominence, being colored a deep 
slaty blue. Two or three methods have been introduced, 



CENTRAL NERVOUS SYSTEM. 1 33 

but the best is undoubtedly the one suggested by Lewis 
(^^Human Brain," p. 125). Sections of the fresh brain 
or spinal cord are cut as thin as possible by means of an 
ether freezing microtome, are then floated on water, and 
afterwards immersed, for the purpose of hardening, in a 
two per cent, solution of osmic acid. After a few minutes 
(about eight to ten) they are washed in water and placed 
in .25 per cent, aqueous solution of aniline blue-black for 
two hours. The sections are once more washed in water, 
and each is spread out on a slide, and allowed to become 
quite dry, after which it is mounted by the addition of a 
drop of Canada balsam and a cover-glass. 

In all the above processes a number of changes are re- 
quired, and if the sections are moved from solution to 
solution by means of needles, they are very apt to be 
damaged, and much time is taken up in carefully manipu- 
lating them. To avoid unnecessary risk, a small perfor- 
ated zinc sieve should be employed, in which the sections 
may be placed, and so transferred altogether from one 
solution to another. 

If it be desired to prepare specimens of the cortex of 
the brain, very good results may be obtained by adopt- 
ing Jolgi's ^'sublimate method." It consists of two 
processes : i. Hardening in bichromate of potash. 2. 
Treatment with bichloride of mercury. Small pieces of 
the tissue are placed in Mliller's fluid for about four weeks, 
and are then passed directly into a watery solution of 
corrosive sublimate. The best strength to use is a .25 
per cent, solution. The volume of fluid must be several 
times that of the specimens, and it must be renewed as 
often as it becomes yellow. An immersion of at least 
three weeks must be allowed, but longer than this im- 
proves the result. Sections are then cut by the freezing 
microtome, and afterwards thoroughly washed in water, 



134 MEDICAL MICROSCOPY. 

Otherwise they will be spoiled by the formation of a black 
precipitate. They are best preserved by mounting in 
glycerine. Prepared in this way the nerve cells and 
sometimes the blood-vessels are opaque and black. Oc- 
casionally the body of the cell and its finest processes 
are outlined with the utmost clearness. 

This is not a true stain, but Jolgi thinks that there is 
formed in the tissue elements a precipitate of some sub- 
stance that renders them opaque. 



CHAPTER X. 

INJECTION OF TISSUES. 

The operation of injecting the blood-vessels is not often 
required for pathological purposes ; but very instructive 
specimens of emphysematous lung may be prepared in 
this way. In the physiological laboratory it is more 
often performed. The process is rather a difficult one, and 
for successful results requires more complicated apparatus 
than a private individual generally cares to set up. 

For the sake of completeness two simple methods will 
be described here, which with a little practice and 
patience will answer very well. Two kinds of '* injection 
masses" are in use, namely, one which is fluid at ordi- 
nary temperatures, and the other which is solid at the 
temperature of the air, but melts by heat ; the latter is 
more generally used. Several varieties of each have been 
introduced, but only two are of importance : — 

Cold Injection. — 

Soluble Prussian blue, . 2 parts, 

Distilled water, loo ^ 

The Prussian blue is dissolved in the water, and a 
few drops of hydrochloric or acetic acid are added be- 
fore injecting. 

Carmine Gelatine Injection Mass. — 

Carmine, •. 3 parts, 

Strong ammonia, 6 *' 

Glacial acetic acid, 6 " 

Coignet's French gelatine, 7 " 

Water, 80 " 



136 MEDICAL MICROSCOPY. 

The gelatine is cut up into small pieces, placed in 50 
parts of the water, and allowed to swell up for four or 
five hours. The carmine is ground up in a mortar with 
a little water, and the ammonia added. This mixture 
is allowed to stand for two hours, and then poured into 
a bottle, the mortar being rinsed with the remainder of 
the water. The swollen gelatine, with any remaining 
water unabsorbed by it, is placed on a water-bath until 
it melts. A dark purple fluid results from the carmine 
mixture, to which the acetic acid is added, a drop at a 
time, the whole being thoroughly mixed ; as soon as the 
color changes to a crimson the addition of the acid 
must be stopped. The crimson carmine is added little 
by little to the melted gelatine, the mass being continually 
stirred. Prof. Stirling states that this mass may be kept 
in a cool place for a long time if its surface be covered 
with methylated spirit. Before it is employed it must 
be melted over a water-bath, and filtered through fine 
flannel, rinsed out in hot water. 

The organ to be injected should be removed immedi- 
ately after death, the main artery being carefully dissected 
out and cut as long as possible. The injections may be 
made wnth a brass syringe, which should be short, and 
wide in the barrel to allow of its being readily cleaned, 
and it should have several nozzles of varying calibre, each 
provided with a shoulder and groove by which to secure it. 

Instead of fixing a nozzle directly on to the syringe, it 
should be fastened into the vessel by means of thread, and 
a stout piece of elastic tubing should be fixed over it, 
connected by the other end to the syringe. When in use 
the nozzle is filled with the injection fluid, as is also the 
syringe with the tubing attached, the latter being closed 
at its extremity by a brass clip, so that all air may be 
excluded; the tubing is then applied to the nozzle, the 



INJECTION OF TISSUES. I37 

clip removed and the piston slowly pushed in, the nozzle 
being steadied by an assistant. A pause must occasionally 
be made to allow the fluid to slowly force its way through 
the vessel. If the blue solution be used no further pre- 
cautions are necessary, but if the carmine injection-mass 
be employed, the organ to be injected must be placed in 
warm water (40° C), and before use the syringe must 
likewise, be heated by drawing hot water up into it several 
times, the injection-mass itself being liquefied over a 
water-bath. 

More frequently injections are made by a *^ constant- 
pressure '^ apparatus. That described by Prof. Stirling 
C Practical Histology," p. 75) is the simplest and best. 
This apparatus consists of a tin trough large enough to 
contain the organ to be injected, and sufficient water is 
poured in to cover it. The water is kept at 40° C. by 
means of a gas burner or spirit lamp placed beneath it. 
In the same trough is placed the injection-mass in a 
Wolff^s bottle. The bottle is connected with a large air 
chamber (a large wide-mouthed jar answers very well) into 
which water can flow from the tap, and thus compress the 
air. The pressure within the chamber can, if desired, be 
registered by means of a manometer. The compressed 
air acts on the surface of the injection-mass in the Wolff's 
bottle, and forces it through a tube which is attached to a 
cannula fixed in the main vessel of the organ. 

After the operation is over, the further treatment 
depends upon which material was used. If the Prussian 
blue solution has been employed, the organ must be at 
once placed in equal parts of methylated spirit and water 
to which a few drops of hydrochloric acid have been 
added ; it is left in this for twenty-four hours, after which 
it may be cut up and hardened in alcohol. 

When the warm carmine mass has been employed, the 



138 MEDICAL MICROSCOPY. 

organ is rapidly cooled by being placed in running water 
until the mass is thoroughly set, when it may be cut into 
small pieces and hardened in alcohol. 



CHAPTER XI. 
EXAMINATION OF FRESH TISSUES. 

It may be often necessary, either in the post-mortem 
room, or especially in the operating theatre, to examine 
a tissue as soon as it is removed from the body for 
purposes of diagnosis. Although the specimens so pre- 
pared are not as satisfactory as when they have been 
submitted to the processes of hardening, and the other 
stages necessary for mounting permanently, yet by means 
of certain precautions very fair results may be obtained. 

Tissues cannot be examined in a dry state on account of 
their opacity and the irregularity with which they transmit 
light. They must, therefore, be bathed in some medium 
which will change their appearances and vital properties as 
little as possible. The examination of blood, pus, effusions, 
etc., will be referred to in future chapters, and we shall 
consider here the treatment of tumors, and the method 
of examining such tissues as nerve fibres, splenic pulp, 
membranes, etc. The fluids usually employed for the 
purpose of moistening such specimens are, normal saline 
solution, serous fluid, aqueous humor, and oxidized serum. 

Normal Saline Solution. — The preparation of this 
liquid was given on p. 41. It can be prepared in bulk and 
kept continually at hand. It alters the tissue but slightly, 
and is the most convenient of all these liquids. 

Serous Fluid. — This is most commonly taken advan- 
tage of in the post-mortem room, where it may always be 
procured from the pericardial sac. 

139 



140 MEDICAL MICROSCOPY. 

Aqueous Humor. — This is obtained by puncturing 
the anterior chamber of the eye of a recently killed ox. 
Iodized Serum. — The usual formula for this is: — 

Tincture of iodine, i part, 

Serous fluid, loo parts, 

Carbolic acid, .5 part. 

Sterling (^^ Practical Histology," p. 24) recommends 
that a strong solution should be kept which may be 
diluted as required. The serous fluid obtained by 
allowing blood to coagulate, and pouring off the serum 
from the clot is placed in a bottle with a few crystals of 
iodine. This mixture must be frequently shaken. At 
first very little iodine is dissolved, but in a fortnight or 
three weeks the solution becomes of a deep brown tint. 
When required, a little of the strong fluid is added to 
fresh serum until the latter has a light brown color. In 
whatever manner prepared, this fluid alters the tissues 
slightly, and colors them a light yellow. 

Thin membranes may be placed at once on the slide, 
spread out as far as possible by means of scissors and for- 
ceps in a few drops of normal saline solution, and a cover- 
glass applied. A better result is obtained if a drop of a 
dilute watery solution of methylene blue is added. The 
stain must be very weak, or it adds to rather than detracts 
from the obscurity of the specimen. 

In the examination of fresh tissues, as a rule, the opera- 
tion of teasing has to be adopted. A small portion of 
the muscle or other fibrous material is cut off with a pair 
of curved scissors and placed on a clean glass slide with a 
small quantity of one of the solutions previously named. 
The elementary parts of the tissue are then separated by 
means of needles. To effect this the particle under exam- 
ination is fixed with one needle, whilst small fragments are 



FRESH TISSUES. I4I 

torn off with the other. These are treated in the same 
manner until they are as minute as possible. The most 
suitable fragment is then placed beneath a cover-glass, the 
bulk of the tissue being removed. 

Another method which may sometimes be adopted, is 
to scrape the surface of the object to be examined, having 
previously moistened it with saline solution, and the scra- 
pings are collected on the slide and examined under a 
cover-glass. 

Great assistance in either of the above processes is ob- 
tained by *' softening" or ** dissociation/' Several 
reagents are used for this purpose, of which the following 
are the best : — 

Iodized Serum prepared as above may be used for 
isolating nerve fibres. Specimens must be allowed to re- 
main in it for thirty-six hours, and the strong iodized 
serum should be diluted until the solution is of a deep 
cherry color. 

A ten-per cent, solution of common salt may be em- 
ployed to soften connective tissue, being especially useful 
in the examination of tumors. 

Dilute Alcohol, according to Ranvier, is very service- 
able for epithelial tissue; twenty-four to thirty-six hours 
are required for its full action. The best strength to use is 
one part of 90 per cent, alcohol with two parts of water. 

Caustic Potash in 40 per cent, solution is recom- 
mended by Woodhead for isolating muscle cells. This is 
very rapidly accomplished, seldom requiring longer than 
half an hour. 

Potassium Bichromate in two per cent, solution is 
useful for dissolving the cementing material between the 
fibres of tendon, or for dissociating epithelium and the 
nerve cells of the spinal cord. It requires seven to four- 
teen days to complete the process. 



142 MEDICAL MICROSCOPY. 

For specimens of the central nervous system, Landois' 
fluid is perhaps as good as any. It has the following 
composition : — 

Sodium sulphate, 5 parts, 

Neutral ammonium chromate, 5 *' 

Potassium phosphate, 5 " 

Distilled water, loo " 

Small pieces of tissue must remain in it from one to five 
days. 

In the investigation of tumors or any solid tissue for 
diagnostic purposes, it is advisable to attempt to procure 
sections. After a considerable amount of practice small 
sections may be cut with a sharp razor. The specimen is 
held in the left hand and short quick cuts are made across 
it, the blade of the razor then being dipped in a glass cap- 
sule containing water. 

The vessel should be placed over a black surface, and 
the cut particles gently moved about with a needle, two or 
three sections will then generally be found thin enough for 
mounting. 

A very useful little piece of apparatus for similar pur- 
poses is Valentin's knife (see Fig. i8), which consists of two 
parallel blades fixed in a handle, which can be approached 
to or separated from one another by means of a screw. 
The tissue to be cut is held in the left hand. The blades 
of the knife are then set almost close to one another, 
moistened with water and drawn with a clean cut through 
the specimen, the resulting section being separated by a 
sharp turn of the instrument, and the blades afterwards 
separated under water. Although this operation appears 
very simple it requires considerable practice before satis- 
factory sections will be obtained; The chief difficulty 
lies in judging the distance at which the blades should be 



FRESH TISSUES. 1 43 

set from one another for various substances. Experience 
alone can teach this accurately. 

The most satisfactory method of obtaining sections of 
fresh tissues is to dip them in gum (see p. 56) and to cut 
the sections by means of an ether-freezing microtome. 
The manner of carrying this out has already been de- 
scribed on pp. 6;^ and 65. The sections may afterwards 
be stained, watery solutions of the dyes being preferable. 
Bismarck brown, magenta and methylene blue may be 
used, and the specimens afterwards mounted in glycerine. 



CHAPTER XII. 

PREPARATIONS OF INDIVIDUAL TISSUES AND 
ORGANS. 

In commencing the study of the microscope in medi- 
cine some difficulty will be probably experienced as to 
which of the foregoing methods is applicable to particular 
tissues and organs. Some hints in regard to this matter 
will be found distributed through the previous chapters, 
but for the sake of easy reference directions will be given 
in this chapter to assist beginners in their choice of hard- 
ening and staining reagents, gathered from the personal 
experience of the author. But after some practice the stu- 
dent will be competent to select his own favorite and suc- 
cessful processes. 

It should be borne in mind that in order to fully under- 
stand diseased organs and tissues the normal structure of 
these parts must be thoroughly known. The student will 
do well, therefore, to examine a number of healthy organs 
obtained from the post-mortem room, or from small ani- 
mals, such as cats, guinea pigs, etc., and in the prepara- 
tion of these considerable proficiency in hardening and 
staining will be acquired. 

We shall begin the list with normal histological struc- 
tures, such as epithelium, and then pass on to the various 
organs. 

Squamous Epithelium. — This occurs either in a 
single layer, or in several layers ; in the latter case it is 
said to be stratified. As a single layer it lines serous mem- 

144 



TISSUES AND ORGANS. 145 

branes — blood and lymphatic vessels, etc., and is known 
as endothelium. A characteristic specimen may be ob- 
tained by killing some small animal, such as a guinea pig 
or cat, and immediately opening the abdomen, removing 
portions of the omentum, or the mesentery, and spreading 
them out on cork. They should then be placed at once in 
a solution of silver nitrate (p. 93), so as to demonstrate 
the outlines of the cells. Other specimens should be 
stained in hsematoxylin or alum carmine. Stratified 
squamous epithelium may be obtained from sections of 
skin, or better, from the tongue of any small animal. They 
may be hardened in absolute alcohol, embedded in celloi- 
din, and stained in picro-carmine or hardened in Mliller's 
fluid, cut by freezing and stained in carmine. 

For the purpose of demonstrating karyokinesis, the spec- 
imens should be stained in safranin, or better, submitted 
to the method described on p. 108. 

Columnar Epithelium. — This form of epithelium 
lines the mucous membrane of the alimentary canal, from 
the cardiac orifice of the stomach, downward, and the 
greater part of the ducts of the glands opening into it. 
Isolated cells may be obtained by scraping the mucous 
membrane of the intestine of some animal, or hardening a 
portion of the intestine in Miiller's fluid, embedding in 
celloidin and staining in hsematoxylin and eosin. 

Secretgry Epithelium. — Typical examples of this 
variety of epithelium may be obtained by scraping the cut 
surface of a liver and examining in glycerine, or by cutting 
sections of a liver, and staining in haematoxylin and eosin. 

Ciliated Epithelium. — This form is best obtained 
from the trachea of any small animal, hardening the speci- 
mens in Miiller's fluid, embedding in paraffin and staining 
in haematoxylin. Very satisfactory specimens may be pro- 
cured from an ordinary nasal polypus by hardening imme- 
13 



146 MEDICAL MICROSCOPY. 

diately after removal in a saturated watery solution of 
corrosive sublimate for two or three hours, then thoroughly 
washing it in alcohol, and staining in hsematoxylin. 

Transitional Epithelium. — This type of epithelium 
is best seen in the bladder ; to obtain specimens the blad- 
der should be spread out on cork, and placed in dilute 
alcohol for twenty-four hours, and stained en masse in 
picro-carmine. A little of the mucous surface is scraped 
off and examined in glycerine. 

Areolar Tissue. — This is rather difficult to obtain. 
The best method is that described by Harris and Power 
('' Physiological Laboratory," p. 92) '- By the injection 
into the subcutaneous tissue of a rat which has just been 
killed, of a 0.2 per cent, solution of nitrate of silver or 
osmic acid, a small artificial bulla is formed. This is 
allowed to remain for from ten to thirty minutes and is 
then opened with a pair of fine curved scissors, and the 
delicate subcutaneous tissue is rapidly removed and 
spread out on a glass slide. It is immediately covered 
with a cover glass, and the preparation is stained for 24 
hours with picro-carmine. Glycerine is passed through 
until all the superfluous staining material is removed, after 
which the preparation is sealed up." 

White Fibrous Tissue. — Typical examples of this 
tissue are found in tendons. Samples may be obtained 
either from the tail of a mouse or rat, or from an ampu- 
tated limb. Preparations may be made by teasing in 
normal salt solution, and staining with magenta, or por- 
tions may be hardened in Miiller's fluid, cut by freezing 
and stained in haematoxylin artd eosin. 

Elastic Tissue. — This is most readily demonstrated 
by teasing out a small piece of the ligamentum nuchae of 
an ox in glycerine, or by mounting a piece of the omen- 
tum of some small animal, washing it freely with dilute 



TISSUES AND ORGANS. 1 47 

acetic acid, staining with haematoxylin and mounting in 
glycerine. , 

Adenoid Tissue (retiform). — This is best exempli- 
fied by hardening a lymphatic gland in Miiiler's fluid, 
cutting by freezing, and staining in haematoxylin. Some 
of the sections should then be mounted directly, and 
others placed in a test-tube half filled with water, and 
thoroughly shaken. This dislodges the lymph corpuscles, 
and leaves the fine reticulum visible. The sections may 
then be dehydrated, cleared, and mounted in balsam. 

Adipose Tissue. — For this purpose specimens of 
skin hardened in Muller's fluid, cut by freezing and 
stained in haematoxylin should be prepared, whilst other 
portions should be treated at once with osmic acid, so 
that the action of this reagent on fat cells may be ob- 
served. 

Gelatinous or Embryonal Tissue. — This form of 
tissue occurs typically in the umbilical cord, and in the 
skin of the embryo. A portion of the cord may be har- 
dened in Mliller's fluid and then in alcohol. Sections are 
cut by freezing, and stained in picro-carmine. Very 
beautiful specimens may also be obtained by forming a 
bulla by the injection of a dilute solution of gold chloride 
into the subcutaneous tissue of an embryo, in a stronger 
solution of which it is subsequently stained (Harris and 
Power.) 

Cartilage. — There are three chief varieties, hyaline, 
elastic, and fibro-cartilage. 

Hyaline Cartilage. — Specimens may be obtained 
from the ribbed cartilages of young animals. They 
should be hardened in chromic acid or spirit, and sections 
cut by freezing, and stained in haematoxylin and eosin. 

Elastic Cartilage obtained from the epiglottis or car- 



148 MEDICAL MICROSCOPY. 

tilages of the ear, should be hardened in spirit and stained 
in picro-carmine. 

Fibro-cartilage. — This variety occurs in the inter- 
vertebral substance. Preparations may be hardened in 
spirit, and stained in the haematoxylin and eosin. Other 
sections should be stained in chloride of gold. 

Ossifying Cartilage. — A long bone of a foetus, such 
as the femur, is decalcified in Perenzi's solution (see p. 53). 
Transverse and longitudinal sections are cut in celloidin, 
and stained in haematoxylin and eosin. 

Bone. — Specimens are decalcified in Perenzi's fluid, 
cut in celloidin and stained in picro-carmine. 

Teeth. — Specimens are placed in a saturated solution 
of picric acid until quite soft, then hardened in alcohol, 
cut in celloidin, and stained in picro-carmine or haema- 
toxylin and eosin. 

In order to show the development of teeth, the jaw of a 
newly-born kitten or puppy should be obtained, and first 
placed in Von Ebner's solution, then hardened in alcohol, 
and stained in lithium carmine and picric acid. 

Striped Muscle. — Any voluntary muscle may be taken, 
hardened in chromic acid and spirit, cut in celloidin and 
stained in picro-carmine. Fresh sections should also be 
examined by teasing in glycerine so as to make oneself 
familiar with unstained muscular fibres, such as are often 
met with in sputum and vomited matters. 

Unstriped Muscle. — The most satisfactory method of 
demonstrating this tissue is by hardening portions of in- 
testine or stomach in Mliller's fluid, cutting in celloidin, 
and staining in haematoxylin and eosin. 

Central Nervous System (see special chapter). 

Brain. — Specimens must be hardened in Mliller's fluid 
or in ammonium bichromate, and cut in celloidin. Sec- 



TISSUES AND ORGANS. 1 49 

tions should be stained in various ways, in carmine, 
aniline blue-black, and by the methods of Weigert and 
Pal. 

Spinal Cord. — The same methods as for the brain 
may be employed. If there is reason to suspect degen- 
eration, Weigert's or Pal's method should certainly be 
chosen. 

Nerve Fibres. — Portions of nerves should be taken 
from recently-killed animals; some of them maybe har- 
dened in Miiller's fluid, cut in celloidin, and stained in 
osmic acid, picro-carmine, or by Weigert's method. 

Others should be placed at once in osmic acid, then 
teased out, and mounted in Farrant's solution. 

Nerve Cells. — In order to demonstrate the different 
variety of nerve cells, specimens of the following should 
be hardened in Miiller's fluid, cut in celloidin and stained 
in aniline blue-black : — 

1. Spinal ganglia. 

2. Sympathetic ganglia. 

3. Spinal cord to exhibit multipolar nerve cells. 

4. Cerebrum. 

5. Cerebellum. 

Nerve Endings — Simple Tactile Cells. — These can 
be well demonstrated in a section of skin, placed immedi- 
ately after removal (<?. g., after amputation) in a solution 
of chloride of gold. Sections are then cut by freezing, 
and mounted in Farrant's solution. 

Pacinian Corpuscles. — To display these bodies, 
sections of skin should be cut, preferably from the foetus, 
hardened in alcohol, and stained in haematoxylin. (For 
further information as regards nerve-endings, see '^ Out- 
lines of Practical Histology," Lesson xxxiv, by Prof 
Stirling). 



150 MEDICAL MICROSCOPY. 

Blood-vessels. Arteries. — The structure of an 
artery is best illustrated by hardening a piece of the aorta 
or carotid artery of a cat in Miiller's fluid, cutting in 
celloidin and staining in picro-carmine. 

Veins. — These may be prepared in a similar manner. 

Capillaries. — These are best obtained from the pia- 
mater. The brain of an animal is placed for two days in a 
2 per cent, solution of bichromate of potassium. The 
pia-mater is then stripped off, stained in haematoxylin and 
eosin, and mounted in Farrant's solution. 

Lfymphatic Glands. — The lymphatic gland of any 
animal may be hardened in Miiller's fluid, cut in celloidin, 
and stained in lithium carmine and picric acid. 

Specimens of sweat and sebaceous glands may be 
prepared in the same way ; the former being taken from 
the sole of the foot, and the latter from the skin of some 
animal. 

The Eye. — The structure of this organ may be de- 
monstrated on the eye of a bullock which has been 
recently killed. It should be hardened in Miiller's fluid 
for three weeks, embedded in celloidin, and the sections 
stained in hsematoxylin and eosin. (For a full account of 
the pathological examination of the eyeball see a '' Manual 
of Clinical and Practical Pathology," by Drs. Wynter and 
Wethered). 

Internal Ear. — The temporal bone of a cat or dog is 
removed and decalcified, and afterwards hardened in 
methylated spirit. It is then embedded in celloidin, and 
sections cut in the direction of the longitudinal axis of the 
cochlea. 

Tongue. — Specimens of this should be hardened in 
Miiller's fluid, cut in celloidin, or by freezing, and stained 
in safranin and in picro-carmine. 



TISSUES AND ORGANS. 151 

Specimens of oesophagus, stomach, and intestine 
should be hardened first in Miiller's fluid, and then in 
absolute alcohol. After embedding in celloidin, the pre- 
parations should be so arranged that sections are cut trans- 
versely and longitudinally to the axis of the intestines. 
Haematoxylin and eosin is the best staining method. For 
the purposes of instruction, sections of the stomach should 
be made through the cardiac and pyloric ends, and from a 
portion of the greater curvature. From the intestine, 
portions should be selected from the upper part of the 
duodenum, the ileum, and colon. 

Lungs. — Portions should be hardened in absolute alco- 
hol, cut in celloidin, and stained in lithium carmine and 
picric acid. The student should make himself familiar with 
the structure of the bronchi and trachea. The process of 
embedding in celloidin is especially useful in examining 
specimens of lungs, more particularly in cases of pneu- 
monia and phthisis. It is almost essential when micro- 
organisms {e, g.y tubercle bacilli) are to be sought for. 

Thyroid Gland. — Specimens are to be prepared by 
hardening the gland for twenty-four hours in a mixture 
of spirit and water, and then in absolute alcohol. Sections 
should be cut in celloidin and stained in picro-carmine. 

Thymus Gland removed from a foetus should be 
treated in the same way. 

Liver. — Hardened in absolute alcohol, cut by freezing, 
and stained in haematoxylin and eosin. 

Sections of Kidney are prepared in the same manner. 

Pancreas and Supra-renal Bodies. — Hardened in 
Miiller's fluid, cut in paraffin or celloidin, and stained in 
haematoxylin and eosin. 

Spleen. — Harden in Miiller's fluid. Place a few sec- 
tions in a test-tube with water and thoroughly shake ; 
then mount the sections unstained, so as to show the struc- 



152 MEDICAL MICROSCOPY. 

ture of the pulp. Other sections should be cut in celloidin, 
and stained in carmine and picric acid. 

Bladder. — The organ should be distended with chro- 
mic acid and spirit, and immersed in the same mixture 
for twenty-four hours. Portions are then cut off and 
hardened in absolute alcohol, and treated in the same way 
as the intestine. 

Portions of the uterus, ovaries, and Fallopian 
tubes should be hardened in absolute alcohol, cut in 
celloidin, and stained in haematoxylin and eosin. Speci- 
mens of testicle may also be treated in like manner. 

Embryological specimens should be hardened in 
Miiller's fluid, cut in celloidin, and stained in haematoxylin 
and eosin. 



CHAPTER XIII. 
THE EXAMINATION OF TUMORS. 

One of the most frequent tasks which a pathologist is 
requested to undertake is the microscopic examination of 
tumors, and as this subject, therefore, comes strictly under 
the head of Medical Microscopy, it has been considered 
advisable to devote a chapter to a brief description of the 
most important growths of this kind. In the operating 
theatre, sections may be made, directly the tumor is re- 
moved, by means of a Valentin's knife, and examined 
either unstained in salt solution (see p. 41), or preferably 
stained by dipping them into a solution of hsematoxylin. 
Their nature can then be generally easily recognized. 
Better specimens can be obtained in a very short time by 
cutting off a small piece of the tumor, placing it for a few 
minutes in gum solution, freezing it on a Cathcart's or 
Swift's microtome, and so obtaining thin and complete 
sections. If permanent preparations are required, the 
tissue must be hardened, stained and mounted by the pro- 
cesses already described. The following classifications 
and descriptions are largely taken from Mr. Frederic S. 
Eve's chapter on tumors, in Mr. Mansell MouUin's 
^* Surgery." 

Although cysts are usually described with tumors, an 
examination of their contents, will be considered in a 
future chapter. 

Fibromata. — These are tumors composed of adult or 
well-developed fibrous tissue. In consistence they vary 
14 153 



154 MEDICAL MICROSCOPY. 

from a density approaching that of cartilage, to a succu- 
lent, yielding, but still not friable tissue. 

A section of the firmer fibromata displays a number of 
fibrous bands standing out on a gray, yellowish, or white 
ground ; or the section is firmly uniform and dull white. 
The fibres either interlace, or are arranged concentrically 
or in parallel lamellae. A scraping yields no juice. Mi- 
croscopically, the firmer tumors are composed of looser 
or more compact bundles of fibres, often wavy and interlac- 
ing or disposed chiefly parallel to each other. Situated on 
the bundles in small numbers, are flattened nuclei, be- 
longing to connective tissue cells, the protoplasm of which 
may be shown to anastomose by processes around the bun- 
dles. The softer forms are made up of interlacing fibrillae, 
more or less thickly studded with large round or oval con- 
nective tissue cells, whose protoplasm is continuous with 
the fibrillae. The chief diagnostic characteristics are then, 
fibrous tissue with scanty cells, and at growing points 
young cells surrounding adult and well developed fibrous 
tissue. 

Myxomata (Fig. 20). — Myxomata are new formations 
of loose, fibrillar, connective tissue, permeated with fluid 
which is rich in mucin. Common examples are polypi of 
the nose, rectum and uterus. 

A section exhibits a glistening, pale, jelly-like tissue, the 
surface of which is raised in the centre. Microscopically 
the tumor is composed of stellate and round cells, of 
which the protoplasm is prolonged in delicate filaments 
which meet one another and form a felting of fibrils. The 
stellate cells are chiefly characteristic of mucous connective 
tissue ; in some growths they exist exclusively, while in 
others the round cells preponderate. The vessels are 
usually abundant and clearly seen, owing to the transpar- 
ency of the tissue ; they often form a wide meshwork. 



EXAMINATION OF TUMORS. 



155 



Enchondromata (Fig. 21). — These are tumors 
composed of cartilage chiefly of the hyaline variety. The 
section is glistening, smooth and translucent, and almost 
invariably shows a number of separate masses or lobes 
divided by bands of connective tissue containing blood- 

FiG. 20. 




Cells from a Periosteal Myxoma of the Thigh [Ziegier). "^ 

vessels. In some cases the section is often greatly modi- 
fied by secondary changes. 

Microscopically, the commoner forms have practically 
the same structure as ordinary hyaline cartilage. In many 



^ Figures 20, 21, 22, 23, 25, 26, from Ziegler's " Pathological Ana- 
tomy," are inserted with the kind permission of Messrs. Macmillan 
&Co. 



156 



MEDICAL MICROSCOPY. 



the cartilage cells are more irregularly distributed and more 
numerous in proportion to the matrix ; the capsules are 
larger, and in growing parts of tumors a single capsule 
often contains two or more cells. The cells at the peri- 



FlG. 21. 



'"''^'->^ 




Section from an Osteoid Chondroma of the Humerus [Ziegler), 

a, cortical layer ; b, medullary spaces or cancelli ; c , periosteal growth ; d, normal 
Haversian canals; e. Haversian canals distended with cartilage, which at 
f contains a core of new bone ; g, cartilage developed from periosteum which 
at h contains bony trabeculae ; i, cartilage developed from medullary tissue, 
which at k contains bony trabecula ; /, original trabecula ; vi, remnants of 
medullary tissue. 



phery beneath the perichondrium are flattened. Not rarely 
stellate ones are observed, especially in the parotid tumors. 
Osteomata. — Of these tumors there are three varie- 



EXAMINATION OF TUMORS. 157 

ties ; the eburnated, compact and cancellous. The ebur- 
nated occur as small flat elevations on the surface of the 
skull. Similar growths also spring from the bones of the face. 

Compact osteomata are observed on the shafts of the 
long bones, with which their structure is identical. 

The name cancellous sufficiently describes the structure 
of the third variety, the most common and widely dis- 
tributed of all. With rare exceptions they are situated 
on the diaphyses of the long bones, near to but not neces- 
sarily over the epiphysial disc ; they are covered with a 
layer of cartilage, the deeper surface of which grows con- 
tinually and is gradually converted into bone. 

It is of course impossible to examine these tumors 
by the methods described at the beginning of this chapter. 
They are, however, more easily recognized by their grosser 
characteristics, their position, etc., than by microscopical 
examination. 

In order to procure sections, it is necessary to place the 
portions which have been removed in one of the decalci- 
fying solutions previously described until all lime salts have 
been removed, when sections may be cut in the ordinary 
way. 

Myo-fibromata. — As the name implies, these tumors 
are composed of muscle and fibrous tissue in varying 
proportions. The muscle presents the ordinary char- 
acters of the unstriped, or involuntary variety, and is dis- 
tributed in inter-lacing fasciculi. The outline of indi- 
vidual fibres is often not easily distinguishable in sections, 
but the fibres may be readily isolated by the process 
of teasing as described on p. 140, chromic acid being the 
best medium in which to perform the operation. The 
naked eye characters resemble those of a coarse fibroma, 
the sections being marked by distinct interlacing fibrous 
bundles. 



158 MEDICAL MICROSCOPY. 

Sarcomata. — This large class of tumors consists of 
growths derived from the connective-tissue constituents of 
the body, but their elements never develop into fully 
formed or adult structures. The sarcomata are divided 
into three chief varieties — the round-celled, spindle-celled, 
and myeloid. Between these many intermediate forms 
are observed ; thus, round and spindle-cells may be 
mingled in nearly equal proportions — mixed sarcoma; 











Fig. 


22. 








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"1. 


















:-^ 


















-^^^i 


c ; 
















-- ^^a> 


V 


















a- 

1 



















Section Through the Margin of a Sarcoma Affecting the Intermus- 
cular Connective Tissue {Zieglei-). 

a, normal muscle fibre ; a^, atrophied muscle fibre ; d, round cells intruded between 
the muscle fibres ; c, fully developed tumor tissue ; d, round cells resembling 
white blood corpuscles. 

and myeloid cells are associated either with round or with 
spindle-cells. Again the spindle-celled sarcomata shade 
off indefinitely into the fibromata. 

Round-celled Sarcomata (Fig. 22). — These again 
are divided into two sub-varieties, the small and the large. 
The former is much the commoner, and its component 
cells are about the size of leucocytes or granulation cells. 



EXAMINATION OF TUMORS. 1 59 

In the latter they are as large as those of squamous epithe- 
lium. A section of a round-celled sarcoma under the 
microscope shows an uniform surface formed of closely 
crowded cells, intersected here and there by a band 
of connective tissue. In this respect and in the character 
of the cells it differs essentially from cancer. The nuclei 
are large and the protoplasm around them scanty ; in fact^ 

Fig. 23. 




^ 



V. 

Section from a Giant- celled Sarcoma showing also Spindle-shaped Cells 

[Ziegler). 

many of the small round-celled tumors appear to be com- 
posed entirely of nuclei, and if the processes described on 
p. 108 are applied to the sections, karyokinesis will be 
demonstrated. The inter-cellular substance varies; it 
is usually scanty and homogeneous, sometimes formed of 
delicate interlacing fibrillae prolonged from the protoplasm 
of the cells, and sometimes of homogeneous bands. The 
blood-vessels are usually abundant, and in many instances 
have no proper wall. 



l6o MEDICAL MICROSCOPY. 

Spindle-celled Sarcomata (Fig. 23) are composed of 
either small or large spindle cells analogous to the fibro- 
blasts of young granulation tissue ; they possess an oval 
nucleus, and their extremities are prolonged into a more 
or less delicate fibre. The cells are closely approximated, 
and are arranged in parallel or intersecting fasciculi or 
bundles. The protoplasm of the cells unites to form a 
coarse stroma. 

Myeloid Sarcomata (Fig. 23) occur almost exclu- 
sively as tumors in the centre of bones, and in the gum as 
epulis. They are characterized by the presence of 
'' giant cells, *' which consist of irregular masses of proto- 
plasm containing many nuclei resembling those met with 
in tubercle. These cells are associated with others, either 
round or spindle-shaped or both. A section has, in parts 
or over the whole surface, a peculiar maroon-red tint, by 
which the nature of the tumor may be recognized. 

Sarcomata connected with bone often undergo partial 
transformation into cartilaginous or osseous material. 
In the former (chondro-sarcoma) the intercellular sub- 
stance is abundant and hyaline. The osteo-sarcomata 
are composed either of round or spindle cells. The 
lime salts are deposited in a delicate fibrous reticulum 
in which the round cells are enclosed. 

Some other varieties of sarcomata have to be men- 
tioned : — 

Myxo-sarcomata. — These occur especially in the 
breast, parotid gland, and testicle. In microscopical sec- 
tions they exhibit, in varying proportions, branched and 
round cells, which stand out distinctly in an abundant 
homogeneous stroma. 

Alveolar Sarcomata. — This variety is much more 
uncommon. It occurs in bone, muscle and subcutaneous 
tissue. The cells are large, round, and not unlike epithe- 



EXAMINATION OF TUMORS. l6l 

Hal tissue. They are collected into alveolar masses bounded 
by delicate bands of fibrous tissue. 

Melanotic Sarcomata. — These originate in moles of 
the skin and in the choroid of the eye. They have a 
blackish color, but the pigment is often distributed in 
irregular masses. In sections they exhibit an alveolar 
structure with a scanty stroma. The cells are usually 
round and large in size ; occasionally spindle cells are 
present. 

The pigment is chiefly deposited within the cells in the 
form of small granules, but some of it will be seen to lie 
free in the stroma. 

Lympho-sarcomata. — This is a variety of round- 
celled sarcoma originating in a lymphatic gland, but never 
involving simultaneously the whole lymphatic system. 
Microscopically round cells are seen arranged in irregular 
cylinders lying in the meshes of a coarse fibrous stroma, 
but no definite reticulum can be discovered. 

Adeno-sarcomata. — This can scarcely be called a 
distinct form of tumor. It consists of sarcomatous tissue 
generally of the round-celled variety, enclosing portions 
of the gland tissue in which it is situated ; thus it is fre- 
quently seen in the breast, parotid glands, and testicle, but 
the gland tissue must not be mistaken for new growth. 

Epithelial Tissue Tumors. 

This is a large and important group, and consists of 
tumors derived from the epithelium of the surfaces and 
glands of the body. There are two main divisions, the 
innocent, comprising papilloma and adenoma, and the 
malignant, /. ^., carcinoma. 

Papillomata. — In the simplest form these are merely 
overgrowths of the normal papillae of the skin or mucous 
membrane. Others are composed of a series of mesentery 



l62 



MEDICAL MICROSCOPY. 



ovegrowths, and some occur as branched masses which are 
seen under the microscope to be covered with columnar 
epithelium (villous growths). 

The external form varies greatly, but in microscopic 
sections they are seen to consist of layers of epithelial cells 

Fig. 24. 




Fibro- adenoma of Mamma [Boyce). 



mainly of the pavement variety, more or less altered in 
shape from pressure. 

Adenomata (Fig. 24). — These are glandular tumors 
and differ in structure according to the gland which they 
attack. The most common variety is perhaps the mucous 
polypi, which are composed of hypertrophied mucous glands. 
In microscopic sections branching tubules are seen which 



EXAMINATION OF TUMORS. 1 63 

are lined with a single layer of columnar epithelium. 
The stroma is soft and made up of fibrillar tissue with 
stellate and round cells. Mr. F. S. Eve {^loc. cit., p. 135) 
says that the statement that adenomata do not secrete is 
erroneous, for the tubules of rectal polypi pour out quanti- 
ties of mucus ; in one specimen of fibro-adenoma of the 
breast he found a quantity of milk which exuded from the 
section. 

Carcinomata. — Cancers are tumors whose structure 
follows the type of the epithelial tissues. 

Anatomically they are divided into groups in accordance 
with the nature of their epithelium. 

Squamous-celled Epitheliomata (Fig. 25). — These 
occur in situations where there is normally a flat epithelium, 
namely, the skin, mouth, pharynx, oesophagus, larynx, 
vagina, etc. In some of the cases the ephithelial pro- 
cesses tend to penetrate into the underlying structures so 
as to produce a more or less diffused infiltration and thick- 
ening of the part. As they grow, these processes soon be- 
come compressed, producing concentric whorls or small 
epidermic globes which are known as cell-nests or birds' - 
nests. The cells as a rule are large, and generally irregular 
in shape owing to pressure. 

Rodent ulcer or cancroid of the skin is also a slowly 
growing form of epithelioma affecting chiefly the face of 
people advanced in years. Under the microscope it is 
'seen to be made up of round or lobulated masses of very 
small, round, or slightly flattened cells. Cell-nests are 
absent as a rule. 

Cylindrical-celled Epitheliomata. — This affects 
parts where the surface is normally covered with cylin- 
drical epithelium, hence it is chiefly found in the 
stomach and intestine. Microscopically, the tumor 
will be seen to be composed of a number of cylinders 



164 



MEDICAL MICROSCOPY. 



and alveoli lined with cylindrical epithelium. The stroma 
is well marked. 

Spheroidal-celled Cancer. — This is the most 
common form of carcinoma. It used to be divided into 
two varieties, hard or scirrhus, and soft or medullary, but 
these terms have lost their pathological significance, the 









h 



a 



^h 




-"^^^l^^^fU- H^^^ ^ ^ — -C®^dCP^Jy^^!^^^^3. 



Section from an Epithelioma of Skin [Ziegler). 

a, epidermis ; b^ corium ; c, subcutaneous areolar tissue ; d, sebaceous gland ; e, 
hair follicle ; _/, cancerous ingrowths from the epidermis ; g^ deep set cancerous 
cell groups; h, proliferating fibrous tissue; z, (above) cell nest; z', (below) 
sweat gland. 



difference depending only on the relative amount of 
stroma and epithelial cells respectively. 

In the hard variety (scirrhus, Fig. 26) the cells are 
small and arranged in elongated groups ; they vary greatly 
in shape and often contain two nuclei. Fibrous tissue is 



EXAMINATION OF TUMORS. 



165 



abundant and dense, so that the walls of the alveoli are 
broad and well n:iarked. The cut surface of the tumor is 
grayish and transparent, with opaque yellow markings 
indicating the existence of fatty degeneration in the cells. 
But little juice can be obtained from the cut surface. 
When placed under the microscope it is seen to contain 
irregularly-shaped cells. 

Fig. 26. 



/-^'^ 




h 


¥ 


d 

e 


J _ 




V 


(t 


, ^ 


c 


|t 




^V^ 




KH^ 




Carcinoma of the Mamma [Ziegler). 

fibrous stroma infiltrated 



a^ stroma ; h, nests ; c, cancer cells ; d^ blood-vessel 
with small cells. 



In the softer tumors (medullary cancers) the stroma is 
scanty and delicate. The cells are large, often coales- 
cent, and arranged in long columns or alveoli. The cut 
surface of the tumor is gray in color, and juice can be 
freely scraped from it. In this juice, cells and free nuclei 
may be found, the latter being large, and mainly oval in 



1 66 MEDICAL MICROSCOPY. 

shape. Carcinomata undergo fatty, mucoid and colloid 
metamorphosis. The fatty change is found in most can- 
cers, especially those of the breast. When the mucoid 
degeneration has advanced (mucous cancer) the tumor 
as a whole is gelatinous in appearance. The change is 
chiefly found in the stroma, the alveoli being bounded by 
broad, transparent, hyaline bands of connective tissue. A 
section presents a uniform, moderately firm gelatinous 
aspect. 

Colloid Cancer is found in growths of the stomach 
and intestines. The epithelium is chiefly affected by the 
metamorphosis, and the stroma only to a lesser degree. 
Small drops of colloid material make their appearance in 
the protoplasm of the epithelium, pressing the nucleus to 
one side, and distending the cell in the form of a vesicle. 
The cell wall gives way, and the cells merge together in a 
gelatinous mass ; to the naked eye such tumors have a 
markedly gelatinous appearance, and on section exhibit 
large alveoli bounded by thin bands of stroma and con- 
taining soft, white, jelly-like material. These tumors fre- 
quently occur as infiltrations penetrating among the con- 
stituents of the tissues. 

If cancer attacks the skin or eye-ball it may become 
pigmented and is then known as melanotic carcinoma, 
but it is a rare form of tumor compared with melanotic 
sarcoma. 



CHAPTER XIV. 

EXAMINATION OF URINARY DEPOSITS. 

In this chapter will be described the microscopic exam- 
ination of urinary deposits. The chemical examination, 
which is even of greater importance, cannot be considered 
here. The reader is referred to larger works, notably 
^^ Clinical Diagnosis, " by Prof. v. Jaksch (translated by 
Dr. Cagney), and ^^ Urinary and Renal Diseases, '^ by Sir 
William Roberts. 

Many morbid conditions of the m*inary tract, however, 
are characterized by a particular deposit, and it is desira- 
ble that whenever disease of the kidney, bladder, urethra, 
or genital tract is suspected, a microscopic examination 
of the urine should be made as well as a chemical ; this 
is especially to be insisted on whenever there is a trace, 
no matter how small, of albumin in the urine, and also 
when there is any suspicion of the formation of a calculus. 

The urine should be collected in a conical glass and 
allowed to stand carefully protected from dust for about 
twenty-four hours, by which time the deposit will have 
fairly settled. It is advisable to add some disinfectant {not 
carbolic acid, which precipitates albumin) to the specimen 
to prevent decomposition. Salkouski's fluid is an excel- 
lent one for the pu4*pose {Deutsche Medicinische Wocheii- 
schrift, xiii, No. i6, 1888). It consists of a fluid contain- 
ing 5 to 7.5 c.c. of chloroform in a litre of water ; about 
2 ozs. of this preparation should be used. A few drops of 
thymol or oil of turpentine also answer satisfactorily. 

167 



1 68 MEDICAL MICROSCOPY. 

When the deposit has finally settled the supernatant 
fluid is carefully poured off. A pipette closed by the 
finger at the upper end is then introduced and some of 
the deposit allowed to enter by momentarily removing 
the finger. On the removal of the pipette the excess of 
fluid around the pipette is removed with a cloth, and 
the tube then allowed to rest for some seconds, standing 
perpendicularly on a glass slide; the heavier particles, 
such as crystals, will thus sink, and in this way a satis- 
factory sample of the deposit is obtained ; a drop or two 
of fluid is then permitted to escape and a cover-glass 
applied. The specimen should then be examined first 
with a half-inch (to detect the larger crystals) and then 
with a quarter-inch lens. 

If the deposit be small, considerable assistance is 
afforded by adding a drop of a solution of iodine dis- 
solved in iodide of potassium (see p. 41) to the fluid on 
the glass slide, or a few drops (three are sufficient) of 
magenta solution to the deposit in the specimen glass. 

The most convenient formula for the magenta is 
Woodhead's: — 

Magenta, i part, 

Rectified spirit, 20 parts, 

Distilled water, 180 " 

By either of the above means, the epithelial cells are 
brought more into prominence, and by their distribution 
in lines, or otherwise, may draw attention to other 
bodies, such as casts of the renal tubes, in which they 
are lying. In addition to this, should amyloid casts be 
present they will be stained a deep brown by the iodine 
solution. 

Another advantage of using a stain is that the objects 
are more easy to focus when some coloring matter is 



URINARY DEPOSITS. I 69 

employed. Unless this be done, the transparent cor- 
puscles, especially if the deposit is small, will often 
escape notice as the lens is gradually approached to the 
glass, with the result that the cover-glass is pushed hard 
on to the slide, and the fluid, carrying crystals, cells, or 
other bodies with it, is forced from beneath the edges 
of the glass and that specimen rendered useless. 

Directions were given above that the urine should be 
allowed to stand for 24 hours before the deposit is ex- 
amined. In certain cases it is also advisable to look 
at a specimen about six hours after it is passed. Some 
urines undergo decomposition very rapidly, or the urine 
may be drawn from the bladder actually decomposed. 
If *^ cayenne-pepper " grains be deposited these should 
also be examined as soon after they crystallize out as 
possible, in order to recognize the uric acid formation ; 
if the urine becomes alkaline from decomposition, these 
crystals are transformed to those of urate of ammonia, 
and thus false impressions may be derived. 

There is generally a slight deposit even with healthy 
urine, and if this be examined will be found to consist of 
epithelial debris, various crystals, and leucocytes. Very 
often also there is a copious deposit of urates, especially in 
the urine passed in the early morning. 

The various objects met with in urinary deposits will 
now be described seriatim, the pathological significance 
of each being duly considered. 

The sediments naturally divide themselves into or- 
ganized and non-organized ; the former including 
epithelial cells, the corpuscles of blood, pus and mucus, 
renal casts, parasites, and portions of new growth ; whilst 
under the latter division will be described the numerous 
forms of crystals which separate from the secretion under 
different conditions. 



170 medical microscopy. 

Organized Deposits. 

I. Epithelium. — As already stated, some epithelial 
cells are found in the very slight deposit which falls in 
healthy urine ; these arise from the urethra and bladder, 
and in females from the vagina, and it is only when 
present in large numbers that their presence is of any 
note ; but a different construction must be put on the 
occurrence of renal cells. 

The cells for the most part are of the squamous variety, 
are polygonal in shape, and contain only one nucleus; 

Fig. 27. 




Vaginal Epithelium, 

distributed amongst them will be found their earlier 
form, circular in appearance. These arise from the meatus 
and prepuce. Those from the vagina are of the same 
shape, but much larger (Fig. 27). 

Bizzozero describes a form of epithelium which has for 
its origin the surface of the male urethra ; the cells are 
cylindrical, have well-marked outlines, and gradually di- 
minish in size toward their attached extremities, and thus 
appear sometimes tailed, at other times rounded, according 
to the surface which is presented to the eye. 

If any of the above cells are present in large numbers, 
some affection, most probably inflammatory, may be as- 



URINARY DEPOSITS. 



171 



sumed, of the parts from which they come, thus, the cells 
described in the last paragraph are especially numerous in 
men suffering from an old gonorrhoea, and Sir William 
Roberts {Joe. cit., p. 122) points out that the deposits 
found in the urine of persons subject to nocturnal emis- 
sions have much the same appearance to the naked eye, a 
collection of whitish flakes and strings being found at the 
apex of the collecting glass. 

In women afflicted with leucorrhoea, large pavement 

Fig. 28. 




Epithelium from the Bladder, Ureter and Pelvis of the Kidney. 



epithelial cells are found in large numbers in the deposit, 
often united by their borders into patches of rude Mosaic. 
The cells derived from the epithelium of the bladder, 
ureter, and pelvis of the kidney (Fig. 28) are difficult to 
distinguish from one another. A great variety of forms 
are found. Bizzozero and Eichorst maintain that the type 
of cells is the same for all these parts ; the cells may be 
spindle-shaped, cylindrical, tailed, spheroidal, oval, or ir- 
regular in shape. They are smaller than those described 
above. Those that come from the superficial layers are 
spheroidal or oval j they possess one nucleus and are 



172 MEDICAL MICROSCOPY. 

generally coarsely granular. The cells arising from the 
deeper layers are more irregular, or spindle-shaped, and 
often present processes projecting from their sides. The 
single nucleus is larger, and the protoplasm is more finely 
granular. In cases of vesical or renal calculi these cells 
are often found in large numbers ; they also occur in 
the urine of patients suffering from pyelitis. 

The recognition of renal epithelium (Fig. 29) is of much 
more importance. The cells are of two chief varieties, 
according as to whether they are derived from the straight 
tubes or from the cortex, but the two kinds are often very 
difficult to distinguish from one another. 

Fig. 29. 




Renal Epithelium, Healthy and Fatty. 

The typical cells from the cortical portion consist of a 
circular nucleus about the size of a red blood corpuscle 
surrounded by a finely granular protoplasm, the outline of 
which is generally indistinct. The cells from the straight 
tubes are flatter and more cubical in shape ; the cell wall is 
generally plainly seen. 

The cells, therefore, are rather smaller than those de- 
rived from the other parts of the genito-urinary tract. 
They are often aggregated together into small groups, or 
form complete casts of the renal tubules (see below). A 
few cells are frequently seen cohering to hyaline and other 
casts. 



URINARY DEPOSITS. I 73 

The presence of these cells indicate some lesion of the 
kidney substance, most probably inflammatory. If the 
cells show fatty degeneration, this is indicative of a similar 
process going on in the kidney, and thus is of grave 
import. 

Prof. V. Jaksch (^* Clinical Diagnosis," p. 179) de- 
scribes another variety of cells which occurs in the con- 
valescent stage of acute nephritis (of scarlatina and ery- 
sipelas). These are small round cells with an eccentric 
nucleus. He considers that they are doubtless the young 

Fig. 30. 



00 



00 

O 



1^^^ 
^ 



Blood Corpuscles in Urine. 

kidney cells formed within the tubules in the process of 
repair. 

2. Red Blood Corpuscles (Fig. 30). — The occurrence 
of blood in the urine must always give rise to some anxiety, 
and a careful clinical examination should be made to try 
and ascertain its source. Much assistance may be gained 
by a diligent microscopical search. 

The presence of blood may be determined by certain 
chemical tests, of which the most satisfactory is the 
guaiacum test. One or two drops of tincture of guaiacum 
are added to a small quantity of urine in a test-tube, and 
about half a drachm of ozonic ether is floated on the 
surface. If blood be present, a blue coloration appears 



174 MEDICAL MICROSCOPY. 

at the junction of the two fluids, which gradually spreads 
through the ether. 

The most reliable test for blood in the urine is, how- 
ever, the discovery of the red blood corpuscles by the 
microscope. The corpuscles do not always retain their 
form and color, in fact, they often appear much changed. 
If the urine be of very low specific gravity, especially if it 
become ammoniacal, the corpuscles gradually lose their 
color, swell by imbibition, and then appear as colorless 
rings and finally may disappear altogether. If, however, 
the urine be of specific gravity 1020-1025, they will 
retain their form, for some days, or appear smaller and of 
a deeper color. Occasionally they shrink and become 
crumpled and misshapen. They occur apart from one 
another and do not run into rouleaux. 

The corpuscles, then, are characterized by their thin, 
clear outline, the absence of a nucleus or cell contents, 
and their feeble refracting power. 

As a rule there is not much difficulty in recognizing 
them, but occasionally other objects occur which by the in- 
experienced may be easily mistaken for blood corpuscles. 

The most likely bodies are the sporules of some fungi ; 
they may be distinguished by having a nucleus, and also 
double forms caused by gemmation may be noticed. These 
sporules also are not always circular, but are often some- 
what oval. 

The nuclei of renal epithelial cells may easily be taken 
for red blood cells ; as already stated, they are of the same 
size and shape and not infrequently the rest of the cells to 
which they belong cannot be made out. This error may 
easily be rectified by the addition of a drop of magenta 
(solution see p. 168) which will stain the nuclei, but not 
the corpuscles. 

The most important point, having proved the presence 



URINARY DEPOSITS. I 75 

of blood, is to attempt to ascertain its source. It may 
arise from the kidneys, renal pelvis, ureter, bladder, or 
urethra. 

As regards the causes, no better classification can be 
found than that given by Sir William Roberts {Jot\ cit., 
p. 141). 

1. Local lesions. — External injury, violent exercise, 
calculous concretions, ulcers, abscesses, cancer, tubercle, 
parasites, active or passive congestion, Bright's disease. 

2. Symptomatic, in purpura, scurvy, eruptive and con- 
tinued fevers, intermittent fever, cholera, etc., mental 
emotion. 

3. Supplementary or vicarious, to menstruation, hgemor- 
rhoids, and asthma. 

That microscopic examination can be of great use in 
determining some of these causes is obvious, thus, small 
crystals of uric acid suggest calculous concretions ; nu- 
merous pus cells denote a possible abscess; tubercle bacilli 
are conclusive proof of a tubercular process somewhere in 
the genito-urinary tract ; the discovery of parasites ; and 
finally the concomitance of renal casts, indicating Bright's 
disease. Some of these points will he again referred to. 

Blood having its source in the kidneys produces certain 
characteristic appearances. The blood is intimately mixed 
with the urine and generally does not form a deposit, even 
after standing several hours, but occasionally" a chocolate- 
colored grumous sediment subsides. The secretion has a 
peculiar reddish or smoky appearance. The cells may 
sometimes be seen to have partially lost their color, and 
present themselves as pale yellow rings, and the lesion 
then is probably an acute nephritis, either of fresh origin 
or an exacerbation of a chronic inflammation. 

If the cells are few in number and have quite lost 



176 MEDICAL MICROSCOPY. 

their color, a suspicion should arise either of miliary 
tuberculosis of the kidneys, or congestion of those organs. 

In cases of abscess, embolism, or tuberculosis, or from 
the presence of parasites, the hemorrhage is usually slight, 
whilst if malignant disease is the cause, it may be very 
frequent and profuse. 

Blood from the pelvis of the kidney and ureter has less 
marked characteristics, and very little help is afforded by 
the microscope. The occurrence of certain crystals, 
such as uric acid or oxalate of lime, may yield suspicion 
of a calculus, but the main evidence must be derived 
from the clinical aspects of the case. The same may 
be said if the blood comes from the bladder or urethra. 
When the bladder is the source, portions of villous growth 
may be found, or there may be other evidences of cystitis. 

If the bleeding is from the urethra, it will probably 
occur independently of micturition. Under either of 
these circumstances the blood will appear of a bright 
red color, will not be intimately mixed with the urine, 
and clots will very likely be found in the deposit; if 
the clots are long and of about the diameter of a goose- 
quill, the ureter is the probable source, as such clots 
are occasionally formed there. 

In the condition known as '' hsemoglobinuria,'' a 
chocolate-colored sediment is deposited. No blood cor- 
puscles are found in it, but it consists mainly of amorphous 
granular matter, which Sir William Roberts considers to be 
disintegrated corpuscles. Tube-casts are also present, mostly 
granular in appearance, accompanied by a few transparent 
fibrinous cylinders. Crystals of oxalate of lime occur, and 
the late Sir William Gull found large numbers of minute 
crystals of haematin. 

3. Leucocytes (Pus Cells), Fig. 31. — A few isolated 
white blood cells will be found in any urinary deposit, 



URINARY DEPOSITS. I 77 

but their presence only assumes importance when there is 
a sufficient number to justify the term '^ pus." The line 
to be drawn between the two conditions is naturally 
an arbitrary one, and no fixed rule can be stated. Gen- 
erally speaking, no conclusions can be drawn from the 
occurrence of the leucocytes, unless there is a distinct 
deposit at the bottom of the collecting glass. 

Urine which contains pus is turbid when first voided, 
but a sediment soon subsides. When the reaction of 
the urine is alkaline, as is usually the case, the deposit 
appears as a semi-gelatinous mass, which is not clearly 
separated from the general mass of the urine, and can 
be drawn out into long strings. Should the specimen 

Fig. 31. 



Pus Cell in Urine Affected by Acetic Acid and Unaltered. 

be acid, the pus forms a uniform white layer, clearly 
defined, but easily miscible with the supernatant liquid. 

The nature of the sediment is generally unmistakable, 
the only deposit likely to be confused with it is mucus. 
Pus may be distinguished from mucus by the addition 
of a little caustic soda, which converts the former into 
a viscid mass, which can be drawn out into long 
strings when the liquid is poured from one test-tube 
into another. 

Under the microscope the pus cells retain their character- 
istic form — spherical in shape, and about one-third larger 
than a red blood cell. They are granular in appearance 
and almost colorless, having only a slight yellowish tinge 
16 



178 MEDICAL MICROSCOPY. 

when crowded together. If the urine is very concentrated 
they appear small and crumpled. The addition of acetic 
acid causes the granules to disappear and the nuclei to 
become prominent. They are often observed to contain 
fatty matter, which indicates that an abscess, of slow for- 
mation, has burst into the urinary tract from some 
neighboring part, and not, as when occurring in epithelial 
cells, fatty degeneration of the kidneys. 

If there be any doubt as to the nature of the cells, this 
may be cleared up by the addition of a drop or two of 
iodine in iodide of potassium (page 41) to a little of the 
deposit on a glass slide j when a cover-glass is applied, and 
the specimen placed under the microscope, the pus cells will 
assume a deep mahogany-brown color, whilst epithelial 
cells and detritus will only be stained a light brownish- 
yellow. The possible source of the pus is the same as in 
the case of blood, namely, the kidneys, pelvis of the kid- 
ney, ureter, bladder, urethra or genital tract, or from the 
rupture of an abscess into the genito-urinary tract from some 
other part. 

Pus from the kidneys is rather rare, according to Sir 
William Roberts {loc. cit., p, ^pp, etseg.), it may arise from 
(i) phlegmonoid inflammations ending in circumscribed 
abscess; (2) multiple abscesses from purulent infection, and 
(3) occasionally from embolism, apart from pyaemia. The 
diiferential diagnosis of these conditions can only be 
arrived at from the. clinical side. With the aid of the 
microscope pus may be assumed to come from the kidneys 
when the pus cells are accompanied by renal epithelium, 
and more certainly when they are accompanied by casts of 
the renal tubes. Occasionally the cells themselves are 
moulded into this form, and are known as ^^pus casts" 
(see below). 

Pyelitis (suppuration in the pelvis of the kidney) is 



URINARY DEPOSITS. I 79 

only productive of a few cells in the urine, and the same 
may be said when the pus comes from the ureter. The 
forPxiS of epithelium accompanying the pus must be carefully 
observed, and from these some conclusions may be drawn. 
With pyelitis the discharge of pus may be intermittent, 
owing to some obstruction blocking back the discharge. A 
tumor, appearing and disappearing, will then probably be 
made out in the loin. An absence of mucus points to the 
mischief being located above the bladder. In the above 
conditions the urine is acid. 

The most common cause of pus in the urine is cystitis. 
The local symptoms will most likely be quite sufficient for 
the diagnosis without the aid of the microscope. The 
urine is strongly alkaline, and often ammoniacal, except 
in old cases^ when it may retain its acidity. The quantity 
of pus is often very considerable. Mixed with the pus cells, 
the numerous forms of bladder epithelium will be seen. 

If the urethra be the source of the pus (as in gonorrhoea) 
the discharge will take place independently of micturition, 
and a small quantity can generally be squeezed from the 
orifice. If pus be found in the urine of a female patient, 
inquiries should be made as regards the existence of 
leucorrhoea before any further conclusions are drawn. 

The bursting of a neighboring abscess is indicated by 
the sudden discharge of a considerable quantity of pus, 
and, as before stated, under these circumstances the cells 
often contain fatty matter. 

4. Mucus. — A translucent, flocculent cloud of mucus 
separates from nearly all urines; when the secretion is 
acid it remains thin and diffluent, but when alkaline, it 
becomes tenacious and ropy. 

There is nothing characteristic to be observed micro- 
scopically, the mucus cells being indistinguishable from 
leucocytes. On account of its common occurrence, it has 



l8o MEDICAL MICROSCOPY. 

little clinical value ; when present in large quantities it may- 
indicate a catarrh of the urinary tract, the exact locali- 
zation of the trouble being indicated by the accompany- 
ing forms of epithelium. 

In order to distinguish a deposit of mucus in acid urine 
from one of pus, the caustic potash test should be 
applied. In the case of mucus, the deposit disappears, 
instead of being transformed into a stringy mass, as with 
pus. The mucin in which the corpuscles lie is precipitated 
by acids and alcohol. 

5. Fat. — Fat globules are but rarely found in urine. 
They may occur as accidental ingredients after the 
passage of a catheter. As a pathological curiosity they 
have been stated to appear after a too excessive or prolonged 
use of cod-liver oil. 

Small globules of fat occasionally accompany casts, 
owing to the fatty degeneration which they have under- 
gone, they also occur in the interior of epithelial 
cells (see page 173), and under similar circumstances 
small globules may be found free in the deposit. In 
^*chylous urine," large quantities of free fat are discharged. 
Globules of fat may then be distinguished under the 
microscope, but more commonly only granular particles 
are visible. 

In phosphorus poisoning also, free fat has been found in 
the urine, which may easily be explained by the intense 
degree of fatty degeneration which takes place in this 
condition. 

6. Casts. — If a specimen of urine is found to contain 
albumin, it has become almost a matter of routine to 
examine the deposit for ^^ casts." This operation is a most 
important one, for all casts (with the exception of hyaline 
casts) are a sure indication of renal disease. Although in 
nearly all cases casts accompany albuminuria, cases have 



URINARY DEPOSITS. l8l 

been recorded in which these bodies were found in non- 
albuminous urine. Thus, Nothnagel ('' Deutsches Archiv 
fur Klin. Med./' xii, 326, 1874) has found them in cases of 
jaundice in which there was no albuminuria, and Drs. Fin- 
lay son {Brit, and For. Med.-Chir. Review^ January, 1876), 
Burkhart and Fischl, have recorded similar cases. The 
form of these bodies indicate that they have been moulded in 
the renal tubules ; their size naturally varies greatly, accord- 
ing to the calibre of the tubule in which they were formed, 
their diameter varying from y^Vo ^^^^ (small) to -^-^ inch 
(large), the largest number being known as '^ medium 
sized, " varying between these limits. 

Their nature is also very different, according to the 
circumstances under which they arise. According to their 
consistence they are known as blood, epithelial and pus 
casts ; cylinders composed of micrococci ; granular, waxy, 
fatty and hyaline casts, the last three often being studded 
with crystals and epithelial cells ; to these must be added 
cylinders formed of crystals and urates, which may be 
known as '^ false casts, " and finally those strange bodies, 
'* cylindroids. " 

There seems to be some doubt as to the precise structure 
of these cylinders. The substratum of most of them is 
apparently an albuminous material exuded from the capil- 
laries, whilst in some cases the casts are formed by 
degeneration and disintegration of epithelial cells and 
blood corpuscles, white and red. This seems to be the 
origin of the granular casts. Blood fibrin is probably 
the basis of the blood casts. Cornil has described hyaline 
globules in the epithelial cells, which are forced into the 
lumen of the tubules, and thus the hyaline variety may be 
found. 

The above objects will be described in the order named, 
together with their clinical significance. 



152 MEDICAL MICROSCOPY. 

Blood Casts (Fig. 32^). — There can be no difficulty 
in recognizing these bodies. The uniform size of the red 
blood corpuscles which usually retain their characteristic 
shape renders the casts conspicuous objects in the sediment, 
more especially as they are generally of the large variety. 
The discs may, however, become crumpled or otherwise 
deformed, which will render their nature somewhat obscure. 
They often appear thick and opaque, owing to the presence 
of a large quantity of dark pigment. These casts are not 
usually perfect, but generally occur as fibrinous casts with 
the corpuscles scattered more or less irregularly over them. 

The discovery of these bodies in a deposit has naturally 
much clinical importance. The most usual condition 
under which they appear is acute nephritis; if sections of a 
kidney affected in this way are examined microscopically 
many of the tubes will be seen to be filled with blood 
and the formation of the casts can easily be imagined. 
Intense congestion, such as occurs after the administration 
of cantharides or turpentine, will also produce them. 
Whether the congestion due to obstructive heart or lung 
disease will cause them to form is open to question. 

These casts are occasionally seen after injuries to the 
kidney in which haematuria results. 

Other rarer conditions accompanied by blood casts are : 
renal calculus, infarct, new growth, and tubercle. 

Pus Casts. — These are but rarely met with even when 
the amount of pus deposited is considerable. 

They appear as cylinders composed of pus cells, closely 
packed together ; they are very rarely complete, and many 
cells will be found lying near them, having become 
detached. It is most likely owing to this latter condition 
that they are so rarely found, as they very easily break up, 
and the majority of them are probably destroyed and the 
cells dispersed before the deposit settles. 



URINARY DEPOSITS. 



183 



The nature of the cells can easily be demonstrated by 
the use of reagents (see p. 178). 

They are most constantly seen in connection with the 
discharge of multiple abscesses into the tubules; conse- 
quently only very few occasions are presented for examin- 
ing them. 

Epithelial Casts (Fig. 32^). — When perfect, these 




a. Blood casts, b. Epithelial casts, c. Granular casts, d. Fatty casts. 
e. Hyaline casts. 

cylinders consist of the characteristic cubical cells of the 
renal epithelium (seep. 172) closely pressed together. Care 
must be taken not to confuse them with blood casts, more 
especially as they occur under similar conditions ; careful 
focusing will exhibit the granular protoplasm surrounding 
the nuclei, and in cases of doubt a drop or two of magenta 
solution should be added to the deposit. As with the 



1 84 MEDICAL MICROSCOPY. 

blood casts, however, this perfect formation is not often 
seen. More commonly the cells are scattered irregularly 
over a hyaline basis. The cell?, too, are generally broken 
or shapeless, or only the nucleus is visible. Immature 
forms of epithelium, oval or circular in shape, are particu- 
larly common. 

The presence of these casts indicate acute changes 
going on in the renal parenchyma, probably nephritis 
or congestion. This condition may either be primary or 
an acute attack upon old mischief; in the latter instance 
they will be accompanied by granular casts. As just 
hinted, cylinders composed of red blood cells and of 
epithelial cells are frequently found together. 

Casts Composed of Micrococci. — These bodies 
are not often met with, and are very likely to be confused 
with finely granular casts. They are markedly opaque, 
and have rather a grayish color. Under a high power 
(y2 immersion) they may be resolved into their individual 
elements and the micro-organisms can be distinctly made 
out. Apart from this they may be distinguished by their 
resistance to caustic potash and nitric acid. 

They imply a very serious condition in the kidney ; 
most probably septic embolism ; but they may also arise 
from the extension upward of septic pyelitis (v. Jaksch). 

Granular Casts (Fig. 32*;). — These are more fre- 
quently met with than any other form. They are 
opaque, usually have a sharp outline, and are gray or 
brown in color. Fragments only of these bodies are 
usually found, and by their uneven extremities show 
further signs of having been broken. There are two 
distinct varieties; in one the granules are so fine as 
only to be differentiated by a very high power, and 
in the other the granules are much coarser, so that they 
can easily be made out with a half-inch lens. 



URINARY DEPOSITS. 1 85 

When present they indicate the later stages of an 
acute renal affection or a more chronic process; and 
naay be said to be diagnostic of Bright's disease. When 
the process is acute they are accompanied by epithelial 
and perhaps blood casts; but when occurring alone, 
contracted granular kidneys may be assumed. No dif- 
ference can be drawn as to the anatomical conditions, 
by observing whether the granules be fine or coarse. 

Most authors now agree that they are formed by the 
degeneration of the blood and epithelial casts. 

Amyloid Casts. — These are clear, homogeneous 
cylinders, highly refracting and possessing a clear outline 
with rounded extremities. They are often of a great 
length, but being brittle, are easily broken, so that 
fragments are more commonly seen than perfect casts. 
They are found of varying diameter, the largest being 
almost an inch in breadth, while the smallest are not 
more than the breadth of a red corpuscle, and between 
these limits all sizes occur. 

Owing to their transparency, these casts are difficult to 
see, and unless some staining solution, such as magenta or 
iodine in iodide of potassium, be employed they are easily 
missed. Attention is sometimes drawn to them by the 
linear arrangement of crystals, leucocytes, or epithelial 
cells clinging to them. 

They occasionally exhibit the amyloid reaction (see 
p. 107) with iodine in iodide of potassium, or methyl- 
aniline violet, but this is by no means constant. 

Their exact composition is not known, but v. Jaksch 
concludes that they are very complex and may result 
from the destruction of epithelium, or from the exudation 
products (amyloid material, fibrin) into the renal tubules. 

They sometimes occur in large numbers. 

Their diagnostic significance is not great, as they are 



1 86 MEDICAT. MICROSCOPY. 

found in all forms of chronic renal disease ; conse- 
quently amyloid disease of the kidney must not be diag- 
nosed from their presence alone. 

Fatty Casts (Fig. 32 d^. — Cylinders composed entirely 
of fatty globules are but rarely met with, although they do 
occur; more commonly the globules are found scattered 
more or less numerously over a hyaline matrix. 

Fatty crystals frequently accompany them, being com- 
posed of crystals of the fatty acids or the combination of 
these with the earthy metals. 

These casts are easily recognized by the sharply defined 
outline and high refracting power of the globules, but 
their nature may be proved by running a drop or two 
of ether under the cover-glass, when the globules will 
be immediately dissolved. 

Fatty casts occur in connection with subacute and 
chronic nephritis, when the disease has lasted for some 
time. They indicate, especially when occurring as pure 
casts formed entirely of fat, an advanced degree of fatty 
degeneration of the kidneys, and consequently necessitate 
a grave prognosis. 

Hyaline Casts (Fig. 32 e), — These are very similar 
in appearance to the amyloid casts already described, but 
are not so highly refractive, and their extremities are 
somewhat more square. They are homogeneous, pale 
bodies, sometimes described as ^^ ground-glass like," and 
are of varying length and thickness. They are even more 
difficult to see than the amyloid casts, and require to 
be stained with some medium, magenta being the best ; 
a few drops of the stain should be added to the deposit 
(p. 168). Again, as with the amyloid cylinders, atten- 
tion is often drawn to them by the arrangement of 
the epithelial cells, etc., adhering to them, and this is 
more marked after the use of the staining material. 



URINARY DEPOSITS. 1 87 

Their diagnostic value is small when occurring alone. 
Henle found them when the kidneys were quite healthy. 
Huppert discovered them in the urine of epileptic patients 
after an attack, no renal lesions being present. 

When, however, epithelial or blood cells are found upon 
them, some conclusions as regards renal disease may be 
drawn. In such cases nephritis may be assumed, more 
or less acute. In fatty degeneration of the kidneys, 
fat cells are often seen thickly crowded together upon 
them. 

Cylinders Composed of Urates, or Crystals 
(*' False Casts"). — When examining a urinary deposit 
rich in amorphous urates, collections closely resembling 
granular casts are frequently observed ; they are caused by 
the accidental accumulation of the urates, and are con- 
sequently not in any way connected with true casts. 
They may often be caused to form by gently moving the 
cover-glass once to and fro with the finger. Unless 
the observer is aware of their nature, they are not at 
all unlikely to be mistaken for casts ; slight pressure 
on the cover-glass will immediately disturb the formation 
and cause them to disappear. Casts composed of hsema- 
toidin crystals have been described, but I have never seen 
them ; they have been stated to occur in the urine of 
infants. 

Cylindroids. — These bodies were first described by 
Thomas in 1870 ('^ Archiv. fiir Heilkunde," xi, 130) and 
are not uncommonly seen in the urine under various con- 
ditions. They are ribbon-like bodies, much longer than 
casts, and are often twisted upon themselves, and studded 
with small crystals and epithelial cells. In general appear- 
ance they are not unlike threads of cotton, but are easily 
distinguished from them by a more irregular outline, the 
absence of fraying at the ends, and the cells and crystals 



155 MEDICAL MICROSCOPY. 

adhering to them in a way which they never do with the 
extraneous ingredients of urinary deposits. 

Thomas first found these bodies in the urine of scarlet 
fever, but they often occur in Bright's disease, more par- 
ticularly in the chronic forms ; in the urine of children, 
too, they are not uncommonly to be seen, and Bizzozero 
has discovered them when there was no renal disease. 

Their origin is doubtful, but they appear to be added to 
the urine after its escape from the kidney. 

Fig. 33. 




Spermatozoa {^Ultzinann), 

Casts of the Seminal Tubes, — These bodies are only 
seen in very rare instances. In general appearance they 
are similar to hyaline casts, but are much broader and 
usually have spermatozoa embedded in them. They are 
occasionally found in the deposit of the early morning 
urine of those patients subject to nocturnal emissions. 

7. Spermatozoa (Fig. ^iZ)- — Spermatic filaments when 
found in urine are seen as pear-shaped bodies possessing a 



URINARY DEPOSITS. 1 89 

minute oval head, not more than y-oi-QTy ^^ch in breadth, and 
a long, tapering tail, slightly thickened at its attachment to 
the head, the entire length being about -g-i-Q inch. They 
are thus easily recognized by a quarter-inch lens. In urine 
they are always motionless, but when examined fresh they 
exhibit active movements. 

In some instances semen escapes with the urine in' such 
quantities as to form a mucus-like deposit, and whitish 
flakes are then often found, which may be removed with a 
pipette and the characteristic spermatozoa demonstrated. 
In medico-legal cases the presence of these bodies may be 
of the utmost importance, especially if found in the vaginal 
mucus. 

In other respects they have very little diagnostic value, 
for many cases are on record in which they were constantly 
found in the urine for years, without any detriment to the 
general health of the patient. This is a matter of some 
importance, as quacks frequently impose upon their victims 
by exaggerating the danger of the occurrence of these 
bodies in the urine, and so reap a bountiful harvest whilst 
administering drugs for the ostensible purpose of averting 
the terrible fate which they predict for those who are un- 
fortunate enough to consult them. 

8. Fragments of Morbid Growths. — In the early 
days of the use of the microscope ^^ cancer cells " were sup- 
posed to be so characteristic as to be easily recognized, 
and consequently great stress was laid upon their detection 
in cases of doubtful diagnosis. Now, however, the term is 
but rarely used, as the cells from a morbid growth differ so 
little from the ordinary transitional epithelial cells that it 
is impossible to distinguish the former from the latter. 

If a great number of large, irregularly-shaped cells be 
found together with a blood-stained sediment, the diag- 
nosis of a malignant growth may be suggested, but this is 



190 MEDICAL MICROSCOPY. 

of no value unless the symptoms and physical signs also 
point toward the same conclusion. 

In some rare cases small fragments of the growth may 
be discharged (especially in females) which are large 
enough to be submitted to a more complete microscopical 
examination, and then the existence of a growth may be 
determined. Those most likely to be met with are por- 
tions of villi from a superficial growth of the bladder, or 
fragments from the surface of a cancerous ulcer of the 
uterus, rectum, or some other portion of the genito-urinary 
tract. 

9. Parasites. — The parasites found in the urine 
naturally divide themselves into pathogenic and non-path- 
ogenic. Every urine when allow^ed to stand will develop 
large quantities of bacteria, their form depending upon 
whether acid or ammoniacal fermentation is set up, but 
these do not necessarily interfere with the search for other 
more important organisms, such as tubercle bacilli and the 
ova of Bilharzia haematobia. 

In collecting the urine intended for the search for micro- 
organisms, the parts about the orifice ot the urethra should 
be carefully cleansed and the secretion be received into a 
perfectly clean vessel ; the importance of these precautions 
is obvious. The deposit should be examined after the 
urine has stood for twelve hours. The parasites having a 
pathogenic value will be first considered, and then those 
which are non-pathogenic. 

Pathogenic Micro-Organisms. 

Tubercle Bacilli. — The discovery of tubercle bacilli 
in the urine is, of course, of great importance, for it indi- 
cates that a tubercular process is proceeding either in the 
genito-urinary tract or its immediate neighborhood. 

After standing for several hours, the supernatant fluid is 



URINARY DEPOSITS. I9I 

poured off from the deposit. A little of the deposit is re- 
moved by means of a pipette, and the outer side of the tube 
is wiped with a cloth ; the open end is then allowed to 
rest for a few seconds on a cover-glass, one drop allowed 
to escape, and distributed over the glass, either by means 
of the pipette or more satisfactorily by placing another glass 
over the first, and gently sliding them apart. Owing to 
the dilution which the bacilli must undergo in the fluid, a 
considerable number of glasses should be prepared before 
a negative answer is given. 

After the glasses are dry they are stained in precisely the 
same manner as will be detailed under the ^^examination of 
sputum." The various steps, however, should be carried 
out as rapidly, yet as gently, as possible, as the film does 
not become very firmly fixed to the glass, and the salts, 
being easily dissolved, may carry some of the other matters 
away with them. 

If no bacilli be found, and yet the other features of the 
case point strongly to a suspicion of tubercle, the follow- 
ing method may be adopted : — 

About 100 c. c. of the urine are mixed with lo drops of 
caustic soda and boiled for five minutes. The mixture is 
then poured into a conical glass and allowed to stand for 
twenty-four hours. Portions of the sediment are then re- 
moved to cover-glasses and examined for tubercle bacilli 
in the ordinary way (Reichert). 

If the result is still negative, it by no means follows that 
the disease from which the patient is suffering is not 
turbercular, but granted that bacilli are found, what con- 
clusions may be drawn ? 

In the first place, as already stated, it is quite certain 
that there is a tubercular lesion somewhere in the genito- 
urinary tract, or in some tissue immediately adjacent to it, 
which is indirectly affecting that tract. In every probabil- 



192 MEDICAL MICROSCOPY. 

ity there is some ulceration, although bacilli have been 
found in miliary tuberculosis where no ulcers could be 
found. 

The character of the predominating variety of epithelial 
cells will lend considerable aid in localizing the lesion. If, 
together with renal epithelium, casts are associated, the 
kidney may be safely assumed to be the organ affected, 
although it is very important to consider most carefully the 
history and clinical facts of the case, and take the micro- 
scopic examination as an aid to diagnosis, rather than an 
infallible test. 

As a rule, the bacilli are very few in number, but occa- 
sionally large colonies are found, but no deductions as to 
the severity and extent of the disease must be made from 
whether few or many rods are seen. 

Erysipelas. —Various authors, notably v. Jaksch and 
Fehleison (^^ Die ^tiologie des Erysipels"), have described 
cases of erysipelas in which nephritis has supervened, and 
in which streptococci have been found in the urine when 
first passed, which are indistinguishable morphologically 
from the streptococcus erysipelatous. This condition 
ceased when the erysipelas disappeared. 

Too much stress must not be laid upon this form of 
bacteriuria, as micrococci have been found in the urine in 
other septic diseases, such as ulcerative endocarditis and 
glanders, so that they have no diagnostic value. 

The spirillum of recurrent fever has occasionally 
been met with, but only when its association with blood 
pointed to the probability of hemorrhage into the kidney 
and the consequent escape of the organisms from the 
capillaries. 

The fungus of actinomycosis has also been stated to 
occur when the urinary organs are attacked. 

Septic Micro-organisms. — These occur in all forms, 



URINARY DEPOSITS. 1 93 

rods and micrococci. They must be distinguished from 
those due to the decomposition of the urine after \i has 
been passed. 

In septic bacteriuria the urine is turbid when passed. 
The most frequent cause of this condition is the use of 
unclean catheters. 

This subject has been most exhaustively studied by Sir 
W. Roberts, and the results of his observations are given 
in his work " On Urinary and Renal Diseases." He 
divides bacteriuria into four groups : — 

Group I. Bacteriuria associated with incipient putrefac- 
tive changes in the urine. — The urine is feebly acid, neutral 
or feebly alkaline, and passes on quickly to decomposition. 
Rods are most frequently seen under the microscope. 
This condition is not infrequent in women suffering from 
leucorrhoea, in men who have suffered from stricture and 
who have frequently used catheters or bougies. 

Group II. Bacteriuria with ammoniac al fermentation of 
the urifie. — This indicates a serious condition. The urea 
is transformed into carbonate of ammonia, the fermenta- 
tion being due to the action of the '' micrococcus ureae '^ 
(Cohn). The organisms occur singly or in chains of two, 
three, or four cocci. This form is apt to arise in old 
stricture cases, in the presence of calculi, after operations, 
in cases of enlarged prostate, and in all other conditions 
in which the bladder is unable to empty itself completely. 

Group III. Bacteriuria without decomposition of the 
urine, — Rods and cocci are found, and the urine, though 
cloudy on passing, becomes clear on standing. The super- 
natant liquid continues transparent and acid for many days, 
and the organisms in the deposit show no signs of multi- 
plying. All this leads to the inference that the seat of 
growth of the organisms is not the urine itself, but some 
portion of the surface of the urinary mucous membrane. 
17 



194 MEDICAL MICROSCOPY. 

The condition is associated with painful and frequent 
micturition and pains about the neck of the bladder. 

Group IV. Aficrococci in the icrine without decomposition. 
— This condition is extremely rare, Sir William Roberts 
having only met with one example. 

Sarcinse. — This seems to be the most suitable place in 
which to mention the occurrence of sarcinae in the urine, 
although they cannot be strictly stated to be pathognomo- 
nic. The organism is discharged with the urine, some- 
times forming a grayish-white deposit. 

In appearance the groups of elements are similar to 
those found in vomit, but are somewhat smaller. The 
seat of their production is probably the bladder. The 
symptoms accompanying its growth are said to be lumbar 
pains and painful micturition. 

Bilharzia Haematobia (Distoma haematobium). 
— The ova of this parasite are discharged with the urine ; 
the worm itself will be described in a later chapter. 

The urine containing the ova is mixed with blood (en- 
demic haematuria) and frequently contains much fat. 

This disease only occurs in hot countries, such as Egypt 
and the Cape, and those cases which have been watched in 
this country have contracted the disease whilst abroad. 
If the urinary deposit be examined, among the pus cells 
and debris will be found minute oval bodies, about yi-g- 
inch long, furnished at the anterior end with a projecting 
spine, while the other is comparativ^ely blunt. 

In the mature ova the embryo may be seen as a granular 
ciliated body. Occasionally the free embryo may be seen 
either rapidly moving about, or slowly stretching the body 
out to its full length and then again retracting it. In 
such cases the empty shell of the ovum will be seen lying 
near the organism. 

Filaria Sanguinis Hominis. — This parasite is more 



URINARY DEPOSITS. 1 95 

easily discovered in the blood than in the urine, and will 
be therefore described in the chapter on ^'The Examina- 
tion of the Blood." In chylous urine this worm may, 
however, be found by examining the deposit in the usual 
way, and will be seen surrounded by blood and pus cells. 
As the animal is viviparous no ova are discharged. 

Eustrongylus Gigas. — This is the largest of the 
nematoid worms, but it is so exceedingly rare in the human 
subject as to merit no more than passing notice. A speci- 
men preserved in the museum of the Royal College of 
Surgeons of London is recorded as having been found in the 
human kidney after death. It is more common in animals. 

In general appearance it much resembles the Ascaris 
lumbricoides, but is considerably larger, reddish in color, 
and possesses six oval papillae instead of three. 

Ascaris Lumbricoides. — This worm is described 
in the chapter on the examination of faeces; it is only 
found in the urine when a communication exists between 
the lower bowel and the renal tract. 

Echinococci. — For a description of the microscopical 
characters of the hydatids and their cysts the reader is 
referred to the chapter on '^The Examination of Dis- 
charges." 

The occurrence of a hydatid cyst in the kidney is very 
rare, but the discovery of the booklets, etc., in the urinary 
deposit may clear up an otherwise obscure diagnosis. The 
condition most likely to be mistaken for this disease is 
hydronephrosis. 

Complete scolices are very seldom seen, as the vesicles 
are broken in the passage down the ureters, and only 
booklets and the portions of the laminated structure of the 
cysts can as a rule be made out. 

The discharge of a cyst is usually attended with blood or 
pus in the urine. 



196 MEDICAL MICROSCOPY. 

With the use of the microscope alone the site of the 
disease cannot be ascertained, and a careful clinical exami- 
nation must be made. The kidney is the most usual organ 
attacked, but the cyst may be situated in or near some 
other part of the genito-urinary tract. 

Non-Pathogenic Micro -Organisms. 

When any urine is allowed to stand for some time it 
soon becomes crowded with micro-organisms. 

Micrococci are usually most numerous, but rods are also 
seen (vibriones) frequently in active movement. The mi- 
crococcus ureae is often found as almost a pure culture on 
the surface of the fluid. 

Moulds are not so common, except on urines which con- 
tain sugar, when they develop to a huge extent. 

If the yeast fungus (Saccharomyces cerevisiae) is found, 
it is a sure sign of sugar in the urine. This fungus occurs 
as oval budding cells, entangled in a mycelium of branch- 
ing threads. 

Another mould which is fairly common is ordinary 
mildew (Penicillium glaucum) ; this appears in acid urine 
which has been allowed to stand. It consists of oval cells 
and mycelia, but aerial hyphae are soon sent off, terminated 
by brush-like filaments arranged in groups, in which the 
spores are contained. 

Infusoria. — Such organisms are never found in fresh 
urine, but only when fermentation has commenced. They 
are similar to those described in the chapter on the 
^^Examination of Faeces." 

Unorganized Deposits. 
Under this head will be described the numerous crystals 
and amorphous collections which occur in urinary deposits. 
The majority of the substances are naturally dissolved 



URINARY DEPOSITS. I97 

in the urine, but are thrown out of solution either on 
account of being developed in excess, or by reason of a 
change in the reaction of the secretion or even of its 
temperature (urates). In addition there are a few addi- 
tional constituents, such as leucin or tyrosin, which depend 
for their production upon some abnormal chemical process 
brought about by disease. 

A conclusion as to the chief nature of the deposit may 
sometimes be drawn from its naked-eye characters. 

Amorphous urates have a characteristic appearance. The 
sediment is loose and powdery, and varies in color from 
a deep red to nearly white, according to the amount of 
urinary pigment carried down with it. A corroborative 
test for urates is the facility with which they dissolve with 
even slight heat; should any deposit remain after warming, 
the sediment was a mixed one ; too much heat must not be 
applied, or albumin may be thrown down. Another 
deposit usually colored is uric acid. The well-known 
*^ cayenne-pepper-like '' grains are seen collected together 
at the bottom of the glass or adhering to its sides. 

If the urine be alkaline, and phosphates be deposited, a 
brilliant white crystalline layer falls to the bottom of the 
glass, and crystals will also be noticed in wavy horizontal 
lines on the sides of the containing vessel. If the sedi- 
ment be shaken up and some of the cloudy urine placed in 
a test-tube and boiled, heat tends rather to increase the 
cloudiness than to diminish it, but the fluid immediately 
becomes clear upon the addition of a drop of nitric 
acid. The above remarks chiefly apply to a deposit of 
triple phosphates. If the urine be alkaline when passed, a 
peculiar pellicle of amorphous phosphate of lime not 
uncommonly collects on the surface of the liquid. When 
shaken, this layer breaks up into small plates, which exhibit 
a play of prismatic colors. 



198 MEDICAL MICROSCOPY. 

The only other deposit which has marked characteristics 
is oxalate of lime. This forms a double layer at the bot- 
tom of the specimen-glass, the upper being well-defined, 
dense, and white in color, while the lower is more like 
a deposit of mucus, loose, and of a gray color. The 
crystals also form lines on the sides of the vessel, as if the 
glass had been finely scratched. 

In examining a deposit for crystals, the remarks made at 
the commencement of the chapter particularly apply. The 
outside of the pipette should be carefully wiped and the 
tube allowed to rest on the glass slide for a short time in 
order to allow the crystals time to fall to the point, and 
then the drop for examination may be allowed to escape. 

These sediments are most easily classified as those occur- 
ring in acid urine and those occurring when the secretion 
is alkaline. 

Sediments Occurring in Acid Urine. 

Uric Acid (Lithic Acid), Fig. 34. — This is usually 
deposited in crystals, but occasionally in a granular form. 
Crystals so minute as to appear as a flocculent cloud are 
occasionally seen after the addition of cold nitric acid to 
urine, and may be mistaken for a cloud of albumin. The 
most common variety is the ^^ cayenne-pepper-like " grains 
already alluded to. 

The primary form of uric acid is a rhombic prism or 
lozenge, but this is so modified as to be hardly recogniza- 
ble, and the number of varieties met with are almost 
countless. If the angles of the crystals become equal, 
quadrangular tables or cubes are produced. Sometimes 
six-sided crystals are seen, much resembling cystin, but 
differing from that substance in having two of their sides 
larger than the others. If the angles are rounded off, 
ovoids or barrel shapes are obtained. At other times rods 



URINARY DEPOSITS. 



199 



are seen, generally aggregated together into stellate or fan- 
shaped masses. One ray is often larger than the others, and 
forms a well-marked spike. Other forms are seen, too 
numerous to mention, but whatever shape the crystals may 
assume they may nearly always be recognized by their 
color. 

The natural color of uric acid is white, but it generally 
carries down with it some of the urinary coloring matter, 
and therefore the crystals are usually reddish-yellow in 
color, varying from a deep orange to alight fawn, accord- 
ing to the tint of the urine. 

Fig. 34. 




Uric Acid. 



The urine is generally strongly acid, but if allowed to 
stand until it is ammoniacal, the crystals seem to be grad- 
ually transformed into urate of ammonia, assuming the 
characteristic globular form ; on the addition of a drop of 
hydrochloric acid they are re-dissolved, and uric acid 
again crystallizes out. It is important, therefore, to ex- 
amine the original crystals soon after they are formed. 

If there be any doubt as to the nature of the deposit, the 
'^ murexide" test should be applied. A little of the sedi- 
ment with a drop of nitric acid is evaporated to dryness 



200 MEDICAL MICROSCOPY. 

on a glass slide over a spirit lamp. To the residue two or 
three drops of ammonia are added. If uric acid be present, 
a fine crimson color makes its appearance, but sometimes 
not for two or three minutes. 

The clinical significance of a deposit of uric acid is 
greatly dependent upon what time elapses between the 
passing of the urine and the appearance of the crystals. 
In perfectly healthy conditions uric acid separates out 
during the acid fermentation, which occurs twelve or 
twenty-four hours after emission. If the sediment occurs 
within four or five hours, it indicates an excess, but has 
still no special pathological importance. It is frequently 
thus met with in gout and chronic rheumatic complaints ; 
in convalescence from febrile affections ; in chronic diseases 
of the heart and lungs which interfere with oxidation of 
the tissues j in certain diseases of the liver ; in strumous 
and tubercular subjects ; in rickets, scurvy, and leucaemia. 

In connection with gout, Sir A. Garrod has shown that 
the blood of a gouty patient contains an excess of uric 
acid, that it is deposited in combination with soda in the 
cartilaginous and fibrous tissues of the joints, and that in 
such a condition the kidneys themselves often suffer. 

It is, however, when the uric acid crystals are deposited 
before the urine cools, or immediately after, that this sedi- 
ment assumes great importance. It may then be feared 
that a similar process may take place in some part of 
the urinary tract, laying the foundation of calculi or 
gravel. Active treatment for the prevention of such 
a calamity is then immediately called for. 

Amorphous Urates (Fig. 35). — This deposit was 
shortly referred to in the general remarks on unorganized 
deposits. 

Urates are naturally colorless, but as deposited in urine 
they are nearly always colored, the tint varying from 



URINARY DEPOSITS. 20I 

orange or purplish to the lightest pink or fawn, but the 
sediment is always deeper in color than the urine from 
which it separates. The deposit is loose, forming a uniform 
layer at the bottom of the glass, and is often accompanied 
by a peculiar film floating on the surface of the fluid. 

The corroborative test is the application of moderate 
heat, which quickly causes the urates to dissolve. The 
chemical nature of the sediment is variable, but it usually 
consists of the mixed urates of potash, soda, and lime 
(rarely of ammonia), the bases occurring in varying 
proportions. Under the microscope minute granules are 
seen but these have no special characteristics. 



Fig. 35.' 
Amorphous Urates. 



The pathological significance of such a deposit is very 
small ; its formation depends upon an acid state of the 
urine and a certain degree of concentration. It is thus 
often observed in a state of perfect health, especially 
durning the winter months and after profuse perspiration. 

In disease it is very frequently met with, especially 
in febrile conditions, when it need give rise to no 
apprehension. 

If urates are constantly deposited, without rise of 
temperature, there is probably organic disease some- 
where in the body, either in the heart, lungs, liver, or 
elsewhere. 
i8 



202 MEDICAL MICROSCOPY. 

Dyspeptic patients frequently complain that their urine 
is thick, especially that passed in the early morning. 

Urate of Soda (Fig. 36). — This is a very rare deposit. 
The specimen from which the accompanying sketch was 
taken, was obtained from the urine of a patient suffering 
from gout. The sediment was yellowish in color, and at 
first glance was mistaken for one of uric acid. Under 
the microscope the characteristic appearances were seen, 
consisting of circular opaque masses, from which spiny 
crystals projected. Some of the globes presented several 
such spines, others only one, variously curved, whilst still 

Fig. 36. 




Urate of Soda. 

Others were destitute of such projections. Some of the 
crystals seemed to be embedded in phosphatic plates. This 
sediment is chiefly found in the urine of children, and 
is deposited before the urine is passed. The spines, 
therefore, are very apt to cause irritation of the mucous 
membrane, and even to collect in masses and block the 
urethra, or form the nucleus of a calculus. 

In rare cases, of which the above is an example, these 
crystals are found in the urine of gouty patients. 

Oxalate of Lime (Fig. 37). — The general character 
of this deposit has already been given on page 198. 



URINARY DEPOSITS. 203 

Under the microscope the crystals are seen to be trans- 
parent regular octahedra, or, especially in albuminous 
urines, they appear as dumb-bell-shaped bodies. 

The octahedra are generally known as ''envelope 
crystals/' this shape being assumed owing to one axis 
of the crystal being shorter than the other two. They 
are seen of various sizes, but not unfrequently the 
crystals are exceedingly minute, and may easily be 
passed over without recognition. Occasionally they 
crystallize as pointed octahedra. The ''dumb-bell" 
forms are in reality flattened, rounded discs, with a cen- 




Oxalate of Lime. 

tral depression on each surface, and according to the way 
in which they lie in the field, appear as dumb-bells, ovoids, 
or circles, and may be made to apparently change their 
shape by a gentle movement of the cover-glass. These 
crystals are soluble in hydrochloric, but insoluble in 
acetic acid. 

Oxalate of lime occasionally falls as an amorphous 
deposit, and can then only be recognized by its solubility 
or more elaborate chemical tests. 

This sediment is frequently found in healthy urines, 
especially after a vegetable diet, and consequently no 
importance can be attached to its appearance, unless 



204 MEDICAL MICROSCOPY. 

it occurs persistently in large quantities, in spite of 
alteration in diet. 

The chief danger is its connection with the fornaation 
of calculi. Much stress, however, has been laid on the 
condition known as ^^ oxaluria " and '^oxalic diathesis," 
by Drs. Prout, Bird, and Begbie, but Sir William Roberts, 
Beneke, and others, have opposed these views, on the 
grounds, firstly, that intense oxaluria may exist per- 
sistently without any of the symptoms attributed to the 
oxalic diathesis, and secondly, that this group of symp- 
toms may exist typically without the occurrence of 
deposits of oxalate of lime in the urine. 

Beneke further shows that oxalic acid has almost its 
sole origin in the azotized constituents of the blood and 
food ; everything, therefore, which retards the metamor- 
phosis of these constituents occasions oxaluria. 

Very little value, again, attaches to the amount of 
deposit which occurs, for the urine may contain a large 
proportion of oxalic acid, without it or its salts being 
thrown out of solution. To ascertain the exact amount 
present, a troublesome quantitative analysis would have 
to be undertaken. 

Sulphate of Lime. — This substance is but rarely 
found in urinary sediments. The author has never met 
with it. Flirbruger and Valentiner have described its 
occurrence in large needle-shaped crystals, and also in 
elongated tablets with abrupt extremities; they are some- 
times arranged in sheaths and rosettes. Amongst them are 
seen masses of indeterminate crystalline structure. Sul- 
phate of lime is also met with in urinary sediments in the 
amorphous form. It is insoluble in ammonia and acids, 
and its detection can only be assured by a rather involved 
, chemical analysis, for which the reader is referred to text- 
books on that subject. 



URINARY DEPOSITS. 205 

The pathological conditions under which this deposit 
occurs have not been ascertained. 

Phosphates. — The various forms of phosphatic deposit 
are met with in feebly acid urines, especially the ^^ stellar 
phosphates " or neutral phosphate of lime, but as they are 
more frequently found in urines having an alkaline reac- 
tion, their description will be postponed until such 
sediments are considered. 

Cystin (Fig. ^S). — Crystals of cystin are in rare cases 
found in urinary deposits. The urine from which it falls 
has generally a sweet-briar-like odor, and is turbid when 

Fig. 38. 




Cystin. 

passed. It is particularly liable to decompose, evolving 
sulphuretted hydrogen, which blackens white glass vessels, 
and the color of the liquid sometimes changes from yellow 
to green. A great part of the cystin dissolved in the urine 
does not crystallize spontaneously, but may be brought 
down by the addition of a few drops of acetic acid. 

To the naked eye the deposit is fawn -colored, very much 
resembling urates. It is not dissolved by heat, and is in- 
soluble in acetic acid, but soluble in ammonia (difference 
from uric acid). When the ammonia solution is allowed 
to evaporate, the cystin separates out as beautiful six-sided 



206 MEDICAL MICROSCOPY. 

crystals, taking the form either of prisms or plates, the 
former being single or arranged in stellate groups. 

To corroborate the presence of cystin in the deposit, 
the urine should be filtered, the sediment on the filter paper 
washed with a little water, and then transferred to a piece 
of platinum foil and heated in the flame of a Bunsen 
burner. Cystin burns with a bluish-green flame, emitting 
white acrid fumes, having an offensive odor resembling 
garlic, but does not melt. 

A spontaneous deposit of cystin, when placed under the 
microscope, exhibits six-sided plates, showing lines 
of secondary crystallization ; they are not often seen singly, 
but are placed on one another, or joined together in long 
strings. They closely resemble one form of uric acid, but 
differ in having all their sides equal. 

Its occurrence in the urine is chiefly of importance as in- 
dicating the possible formation of gravel or a calculus. It 
has a peculiar tendency to run in families, especially affect- 
ing children and young male adults. Cystin may persist in 
the urine for many years, perhaps only appearing at 
intervals, without any injury to the general health, but 
merely causing physical irritation to the bladder and ure- 
thra by the formation and passage of small concretions. 

Xanthin. — Bence Jones, Douglas Maclagan, and Budd 
have described this substance in urinary deposits. Bence 
Jones discovered it in the urine of a lad who three years 
previously had exhibited the symptoms of renal colic. 
The crystals resemble one form of uric acid, being pointed 
ovals, very much like whetstones. They are soluble in 
ammonia and insoluble in acetic acid (difference from uric 
acid). When the deposit is heated with nitric acid, a 
yellow mass is deposited on evaporation, which turns a 
violet-red color when caustic potash is added. The clinical 
significance is not known, beyond indicating the possible 



URINARY DEPOSITS. 207 

formation of a calculus composed of this substance, of 
which there are now several on record. 

Tyrosin (Fig. 39). — Together with leucin, the crystals 
of which will be immediately described, this substance has 
been found in urinary deposits. 

It occurs as sheaves of very fine needles, sometimes in 
yellowish-green crystalline globules. 

If the nature of the sediment is doubtful, it should 
be dissolved in hot ammonia, and the following tests 
applied : — 

Fig. 39. 




Tyrosin. 

1. If a little of the solution be allowed to evaporate, 
tyrosin will crystallize out in fan-like groups of colorless 
needles. 

2. Some of these crystals are dissolved in hot water, and 
to the warm solution some nitrate of potash and mercuric 
nitrate are added. A red precipitate, together with a red 
coloration of the fluid, indicates the presence of tyrosin. 

3. Some more of the crystals are placed in a small 
capsule and a drop or two of sulphuric acid added. 
After standing for half-an-hour water is added. The 
solution is now boiled and saturated with carbonate of 
lime. The mixture is filtered, and to the filtrate a little 



2o8 MEDICAL MICROSCOPY. 

perchloride of iron solution (which must be free from acid) 
is added. In the presence of tyrosin a deep purple tint is 
produced. 

A spontaneous deposit of tyrosin is rare, but it may be 
obtained from urine which contains it by the addition of 
acetate of lead, until a precipitate is no longer produced, 
and then passing sulphuretted hydrogen through the mix- 
ture. The precipitate is separated by filtration, and the 
clear solution which passes through concentrated by 
evaporation, when the tyrosin will crystallize out. 

This substance is principally found in the urine of 
patients suffering from acute yellow atrophy of the liver, 
but has also been met with in phosphorus poisoning, typhus 
and other infectious fevers, also in leucocythsemia and 
certain nervous states. 

Leucin (Fig. 40). — As an urinary deposit this substance 
is still more rare than tyrosin, although it usually occurs 
in solution with that substance in the above-named con- 
ditions. 

The sediment, when it occurs, consists of yellowish oily- 
looking drops, which have a concentric appearance. The 
urine should be evaporated to dryness, and the residue 
heated with boiling alcohol. On cooling, the solution 
deposits shining, white, delicate plates of leucin, which are 
greasy to the touch ; they are insoluble in ether and 
chloroform (difference from cholesterin). 

As a confirmatory test, some of the crystals may be 
dissolved in boiling water, and the solution heated with 
boiling proto-nitrate of mercury, when, if leucin be pres- 
ent, metallic mercury will be deposited. 

Hippuric Acid. — Crystals of hippuric acid appear as 
'colorless, four-sided, rhombic prisms or needles, the 
former forming when it separates from a dilute^ and the 
latter when from a strong solution. They are soluble in 



URINARY DEPOSITS. 209 

alcohol and insoluble in acetic acid, thus differing from 
uric acid and phosphates respectively. 

They have no clinical significance. They occur in 
large numbers after a patient has been taking benzoic acids 
or after eating certain fruits. 

Hsematoidin and Bilirubin. — These substances are 
both found in urine, but it is extremely difficult, if not 
impossible, to distinguish them one from another. Their 
crystals have the same form, and no accurate chemical 
tests have been discovered which one will react to and the 
other not. The crystals are of two kinds, clusters of nee- 
dles or minute rhombic tablets. Their color varies from 




a light yellow to a deep ruby red. They are soluble in 
caustic soda. On treating the crystals with a little nitric 
acid, a green rim forms round them, and their red color 
gradually changes to blue (Hoppe-Seyler). 

These two substances occasionally occur in the amor- 
phous form. 

Haematoidin crystals have been found in severe acute 
nephritis, in carcinoma of the liver, in some of the acute 
specifics, such as scarlet fever and typhoid, and in jaun- 
dice. If they occur in large numbers, a previous hemor- 
rhage is indicated, or an abscess may have burst into the 
urinary passages. 



2IO 



MEDICAL MICROSCOPY. 



Bilirubin has chiefly been discovered in the urine of 
jaundice. 

Soaps of Lime and Magnesia. — In the urine of 
various diseases, v. Jaksch (Joe. ctt., p. 201) has found crys- 
tals similar in form to those of tyrosin, forming rosettes of 
fine needles. Their nature is uncertain, but this observer 
considers that they are probably composed of the lime and 
magnesia salts of the higher fatty acids. They usually 
occur in the urine of febrile conditions, such as severe 
puerperal septicaemia. 

Sediments Occurring in Alkaline Urine. 

Triple ^Phosphates (Fig. 41). The most typical 
form in which the phosphate of ammonia and magnesia 

Fig. 41. 




Triple Phosphates. 



(MgNH^POi + 6Aq.) occurs in urinary sediments, is a 
triangular prism with beveled ends ; but this is often con- 
siderably modified by the removal of the angles or the 
hollowing-out of the sides. Some of the crystals are more 
quadrilateral in form, whilst others appear almost like 
octahedra in consequence of the central part of the crys- 
tals not being developed. If the urine be highly ammonia- 
cal, the crystals may have a stellate arrangement, consisting 



URINARY DEPOSITS. 211 

of feathery rays, like snow-flakes, or may form peculiar 
jagged figures like leaves. 

Like all phosphatic deposits, these crystals dissolve 
readily in acetic acid. 

They cannot be said to possess any pathological value, 
their presence simply indicating an excess of alkalinity of 
the urine. 

Neutral Phosphate of Lime (Stellar Phosphates), 
Fig. 42. — The chemical formula of this substance is 
CaHPO^ + 2Aq. The crystals are formed of rods or 
needles varying in size, often showing lines of secondary 

Fig. 42. 




f 

Stellar Phosphates. 

crystallization, and arranged singly or in sheaths and 
rosettes. They are generally surrounded by small crystals 
of oxalate of lime. The urine is, as a rule, feebly acid, 
or just becoming alkaline. The deposit is decomposed 
by ammonia and dissolved by acetic acid. This sediment 
is rarer than the other forms of phosphates and has a 
rather more grave significance. Sir William Roberts 
states that, according to his experience, '' the presence 
of this deposit in quantity is an accompaniment of some 
grave disorder." The author has twice met with it in 
cases of advanced phthisis. 

In scanty numbers the crystals may be met with in 



212 MEDICAL MICROSCOPY. 

healthy urine, especially during the ^'alkaline tide" after 
meals, and if the secretion be rich in lime. 

Basic Magnesium Phosphate. — These are likewise 
but seldom met with, chiefly owing to its easy solubility. 
The crystals occur as strongly refracting plates, having the 
form of elongated rhomboidal tablets. 

Nothing is known as regards their pathological value, but 
they simply seem to indicate that the urine is concentrated. 

Amorphous Phosphate of Lime. — This is the 
most common deposit when the urine is alkaline from 
fixed alkali j crystals of the triple phosphate often ac- 
company it. 

In general appearance the sediment is not unlike the 
ordinary urate deposit, except that it is not colored by the 
urinary pigments, and is consequently whiter than the 
supernatant liquid. 

Under the microscope minute granules are seen, 
resembling the amorphous urates, collected together into 
irregular groups. 

Such a deposit is frequently seen in healthy urine 
after a meal, and is a sure sign that the secretion is 
alkaline, and thus is a ready test of the success of drugs 
which have been administered in order to secure an 
alkaline reaction of that fluid. The urine is turbid when 
first passed, and the deposit rapidly subsides, an iridescent 
film collecting on the surface. Amorphous phosphate of 
lime (bone-earth) forms a large portion of phosphatic 
calculi, but requires some nucleus around which to ac- 
cumulate. 

Carbonate of Lime. — This substance is always de- 
posited in the amorphous form from amnion iacal urine. 
The granules are often collected together into dumb-bell- 
shaped masses or small concretions. The sediment dis- 
solves in acetic acid with the evolution of gas, thus distin- 



URINARY DEPOSITS. 213 

guishing it from the amorphous phosphatic deposit which 
dissolves in acetic acid without the evolution of gas. 

When voided as gravel or small calculi, carbonate of 
lime is found in the crystalline form as small spheres ex- 
hibiting a radiating appearance. 

Urate of Ammonia. — Urate of ammonia is thrown 
down when the urine becomes strongly ammoniacal. 
The deposit may easily be obtained by allowing urine 
which contains the amorphous urates or uric acid to stand 
until ammoniacal fermentation commences. 

To the naked eye the deposit is white, but occasionally 
it possesses a beautiful violet hue (Roberts). 

Under the microscope, dark, opaque, globular masses of 
various sizes are usually seen. More rarely this substance 
takes the form of minute dumb-bells, arranged singly or in 
groups. 

If the deposit be dissolved by the addition of a drop of 
acetic acid, and a few drops of the solution placed on a 
glass slide, rosettes of uric acid will crystallize .out, the 
crystals being quite colorless. 

Cholesterin (Fig. 43). — The occurrence of cholesterin 
in urine was first pointed out by Dr. Lionel Beale (^^Micro- 
scope in Medicine," p. 355). This author showed that the 
oily particles so frequently seen in the deposits from the 
urine in chronic Bright's disease contain cholesterin, which 
may readily be separated in a crystalline form. 

But as a spontaneous deposit, cholesterin is extremely 
rare. It has been found associated with pyuria. The 
crystals take the form of rectangular plates with a notch in 
one corner, and commonly appear superimposed one on 
another. 

Indigo. — This substance occurs either as the coloring 
matter of deposits, such as urates, or pure, in the amor- 
phous or crystalline form. The crystals are usually needle- 



214 



MEDICAL MICROSCOPY. 



shaped and arranged in rosettes. It not infrequently occurs 
in putrescent urine as blue shreds and films on the surface 
of the liquid or adhering to the sides of the glass. The 
presence of indigo is due to the decomposition of indoxyl. 

It has no decided clinical significance. 

Gravel and Calculi. — The composition of these bodies 
is more easily determined by chemical analysis than by the 
use of the microscope. 

Reference has already been made several times, whilst 
describing the various deposits, to the possible formation 

Fig. 43. 




Cholesterin. 



of calculi. Thus gravel is most usually composed of uric 
acid or of urates, or of the two combined, more rarely of 
cystin or xanthin. 

Calculi may be formed of the mixed phosphates, carbon- 
ate of lime, phosphate of lime, uric acid, urates (chiefly 
found in the kidneys of children), oxalate of lime ^ mul- 
berry calculi"), cystin, or xanthin. Various other concre- 
tions are occasionally met with, formed of fibrinous ma- 
terial, blood, fatty matter, or cholesterin. 

As regards the microscopical evidence which may be 
obtained from scrapings of these bodies, reference must 



URINARY DEPOSITS. 215 

be made to the different heads under which the crystals 
are described. But as such scrapings will, in most cases, 
merely yield an amorphous powder, but little aid is ren- 
dered in the recognition of the stones, and it is therefore 
advisable to proceed at once to a chemical analysis. 

Extraneous Matters. — Matters entirely foreign to 
the urine, which occasionally find their way into the urine, 
are of two classes, the first of which is of great importance, 
whilst the second is purely accidental. 

In the first class are included such substances as are the 
result of disease, or of a morbid process in some adjacent 
organ. Thus, substances belonging to the faeces may be 
found under such circumstances that they could not have 
been passed by the bowel, thus indicating a communi- 
cation between some point of the urinary tract (most 
probably the bladder) and the intestine. 

In other cases hairs have been passed, which, by the 
malignant course of the case, have been shown to come 
from a dermoid cyst. Fragments of tumors also occur, 
which have their source, not in the urinary organs, but in 
some neighboring tissue. 

The second class of extraneous matters includes any sub- 
stances which have accidentally found their way into the 
urine, or, in some hysterical cases, have been purposely 
placed there for the purpose of deception. 

It is always advisable, therefore, to cover specimens 
which have been set aside for examination, for otherwise, 
in hospitals especially, many small particles derived from 
the bedding, floors, food, etc., may fall in and be a source 
of much perplexity before their true nature is ascertained. 

One of the most puzzling of these objects is caused by 
the use of dusting powders, especially in female patients. 
Starch grains are then seen, more or less altered by imbi- 
bition of fluid, and may be rather difficult to recognize ; if 



2l6 MEDICAL MICROSCOPY. 

a drop of iodine solution is added, however, their change in 
color to a deep purple will at once explain their nature. 

Fragments of pine wood from the floor, becoming soft 
and swollen by soaking, often give the appearance of casts 
unless carefully examined. 

Other substances encountered, and with which the ob- 
server should make himself acquainted, are hairs, both 
human and from animals (especially cats and dogs), fibers 
of cotton, worsted, silk, wool, etc., these being often col- 
ored ; portions of feathers ; bits of tea-leaves, showing cells 
and spiral vessels ; particles of food, meat fibers, oil glo- 
bules, etc. Young pediculi, and the larva of the blow-fly 
(these last especially in diabetic urine) are also sometimes 
found, and hysterical people will add flour, sand, and 
other powders, milk, or blood from the finger, in order to 
endeavor to deceive the physician and excite sympathy, 
generally of a practical form, for some pretended malady. 

Preservation of Urinary Deposits. — A question 
often asked is: ^* What is the best mode of making per- 
manent preparations of urinary deposits ? " This applies 
more especially to the various forms of crystals. The or- 
dinary mode of mounting with Canada balsam is not sat- 
isfactory, this medium being too transparent for the 
purpose. 

The best plan is to use some preservative fluid in which 
the crystals are not soluble. For uric acid, oxalate of 
lime, urates of soda and ammonia, as well as for casts, 
glycerine, diluted with water, answers as well as any, or a 
dilute solution (i in 50) of carbolic acid may be used. 

The method introduced by Dr. Lionel Beale is an ex- 
cellent one and is carried out as follows (^* Microscope in 
Medicine," p. 371): — 

A shallow ^' cell " has to be prepared. The most con- 
venient form for the purpose is that which is made by 



URINARY DEPOSITS. 21 7 

painting upon a glass slide, with a fine brush, a narrow 
border of Brunswick black, enclosing either a square or 
circular space, as may be most convenient. 

The urinary sediment is allowed to subside to the bottom 
of a conical glass, the supernatant liquid being poured off, 
and a small quantity of the preservative solution added.' 
The deposit is again allowed to subside, and the solution 
poured off and replaced by a fresh quantity. This method 
is adopted in order that the deposit may become thoroughly 
saturated with the preservative fluid, otherwise there is 
danger of the preparation deteriorating before many 
months have passed. Glycerine, if used, must be added 
to the deposit in the conical glass in very small quantities 
at a time, so that cells, casts, and other small bodies may 
swell out again after they have been caused to shrink. 

After the final subsidence of the deposit, a small portion 
may be removed with a pipette, placed in one of the cells 
above described, and the glass cover applied to the surface 
of the liquid, care being taken that the whole surface of 
the glass is well wetted with the solution, in order that no 
air bubbles may be included in the preparation. Any 
excess of fluid is now to be soaked up with a clean cloth 
or filter-paper, and the cover cemented to the cell by 
applying a little Brunswick black or Dammar varnish 
with a camel's hair brush. 

Crystals of the triple phosphate or cystin have to be 
preserved in a different manner. The cell is made as 
before. The triple phosphate may be kept in water, to 
which a little ammonia and chloride of ammonium have 
been added. In this solution the crystals preserve their 
beautifully smooth character, while in pure water the sur- 
face becomes roughened. 

Crystals of cystin are best preserved in organic acids, a 
very weak solution of acetic acid keeping them quite 
unchanged. 
19 



CHAPTER XV. 
EXAMINATION OF THE F^CES. 

Under the term ^^ faeces" will be understood all those 
substances passed by the bowel, and consisting of the 
residue from the processes of digestion, mixed with the 
products of the secretions of the alimentary canal. 

In this chapter will be considered, not only the matters 
which are expelled as the result of alterations in the dejecta 
produced by disease, but also objects such as parasites, 
which are added to or passed independently of the stools. 

Before passing to the microscopical examination, a short 
epitome will be given of the chief alterations in the macro- 
scopical characters of the faeces. 

The character of the stools in health is greatly dependent 
upon the nature of the food which is taken, as will be seen 
by the following remarks. 

No definite conclusions can be drawn from the quantity. 
The average in health is about five ounces daily, any con- 
siderable increase being principally due to the addition of 
fluid, which, however, bears no relation to the quantity 
taken by the mouth during the day. Naturally, the quan- 
tity varies considerably with the solid food that has been 
eaten. 

The color changes more than the other characteristics. 
In this respect the nature of the food has a marked influ- 
ence. The usual brown color becomes lighter with milk 
diet, and darker when much meat has been taken; and 
the same effect is produced when the faeces have been 

218 



EXAMINATION OF THE FyECES. 219 

retained for some time in the intestines. An excess of 
green vegetables also communicates their color to the 
dejecta. Cocoa-nibs or chocolate often cause the stools 
to assume a grayish tinge. Coffee, claret, and porter, 
especially when these liquids are taken in excess, give 
their particular hues to the contents of the intestine. 

The color is further considerably altered when various 
drugs have been administered. Iron, mercury, manganese, 
and bismuth cause a black coloration, owing to the forma- 
tion of the sulphides of the metals. Calomel yields a 
green, rhubarb, santonin, or senna a yellow, and logwood a 
red color. 

Various diseases produce such changes in color as to 
almost render them characteristic. The stools are almost 
colorless in obstructive jaundice, when they have a pale 
grayish, putty-like tint. In obstruction of the pancreatic 
duct, too, the stools are almost white, this being due 
to the fact that the action of the pancreatic secretion 
is essential to the production of the ordinary faecal col- 
oring matter (Walker, ^* Trans. Roy. Med. and Chirurg. 
Soc," Vol. Ixxii, 1890, p. 257). 

In the disease known as psilosis or sprue, the motions 
are pure white, with occasionally a layer of yellowish 
matter over them; they are partially or wholly formed 
according to the stage of the disease. The ^^ rice-water " 
stools of cholera are also almost colorless, but are more 
characterized by the consistence. In typhoid fever the 
motions are the color of pea-soup. The occurrence 
of blood in the evacuations, on the other hand, produces 
a black color (melsena), if it is retained for some time in 
the bowel, but if due to the bursting of a vessel through 
ulceration it retains its red color. 

In dysentery the stools vary considerably with the 
different stages of the disease, being sometimes merely 



2 20 MEDICAL MICROSCOPY. 

Streaked with blood, at others consisting of almost pure 
blood, whilst sometimes they resemble meat- washings. 

Bile pigment not unfrequently makes its appearance, 
and is always pathological, depending upon some change 
in the liver or gall-bladder. 

Green stools are principally seen in intestinal affections 
of children. As already stated, this may be owing to calo- 
mel having been administered, but apart from this they 
may occur under two other conditions. Firstly, in 
infants diarrhoea is easily caused by excess of acidity, 
and the green color which is then observed is caused by 
the presence of a bacillus (Lesage), which can be cultivated 
on artificial media, and causes blood poisoning when in- 
jected into animals. Secondly, the condition of green 
stools in children is sometimes brought about by the 
presence of biliverdin. 

The consistence of the faeces depends partly upon the 
food which has been taken, but chiefly upon the quantity 
and nature of the secretions of the large intestine. 

The reaction is normally alkaline, but an acid reaction 
does not necessarily imply disease. In young infants errors 
in feeding frequently produce acid stools, with diarrhoea 
and excoriation of the buttocks, whilst the ^* clayey " stools 
are usually alkaline, owing to the presence of carbonate of 
ammonia, the result of decomposition. 

The odor of the motions yields little information. In 
children, a fetid or acid smell often indicates that they are 
not being fed properly. The absence of bile, by with- 
drawing its deodorizing action, causes a very bad odor. 
In sprue, the smell is almost characteristic, being particu- 
larly pungent and penetrating. 

In stools of dysentery and melaena, and in any condition 
in which decomposition sets in rapidly, the odor becomes 
excessively fetid. 



EXAMINATION OF THE F^CES. 221 

Passing next to objects visible to the naked eye and not 
normal constituents of the faeces, we may mention first 
foreign bodies. These are of all kinds, and the case- 
books of the lunatic asylums record many extraordinary 
things which have been swallowed and afterward passed by 
the bowel. 

Among the most common of such bodies are portions 
of indigestible food, as seeds and stones of fruits, false 
teeth, masses of hair, small stones, and portions of metal, 
etc. When there is over- action of the small intestine 
(lientery) undigested food is nearly always found in con- 
siderable quantities. 

In order to separate these and the following objects, the 
faeces should be placed in muslin over which a stream of 
water should be directed ; by these means most of the 
solid matter will be broken up and the larger bodies 
easily detected and separated. 

Mucous and Fibrinous Shreds. — Nothnagel ('' Beit- 
rage zur Physiologic und Pathologic des Darmes,*' p. 185) 
has described an affection of the bowel which he has termed 
^^ enteritis mucosa or membranacea." In this disease cy- 
lindrical shreds of mucous membrane, sometimes forming 
complete casts, are passed by the rectum, accompanied 
with violent straining. After retention in the lower part 
of the bowel, they may be consolidated into ^^hard, white, 
rounded masses, about the size of nutmegs '^ (Fagge). Dr. 
Lionel Beale ('* Microscope in Medicine," p. 291) has 
described several such cases. Some of the shreds seemed 
to consist of firm mucus, in which the epithelial cells from 
the large bowel and mucous corpuscles were embedded. In 
some of the cases the substance passed was firm enough to 
form a tough, leathery membrane, and under the micro- 
scope exhibited fibres, mucous corpuscles, and imperfectly 
formed epithelial particles. 



22 2 MEDICAL MICROSCOPY. 

Occasionally the casts are more distinctly fibrinous and 
are tougher and ribbon-like ; their form and character can 
be demonstrated by removing them to a large dish of water 
and gently moving them to and fro with a needle, when 
they will float out and exhibit their whole structure. On 
careful examination pits may be made out corresponding 
to the mouths of glands. 

Portions of Bowel Separated from Intussus- 
ceptions. — These are more interesting from a purely 
pathological than from a clinical point of view, as the 
diagnosis of intussusception will in all probability have 
been made before the cast-off portions are passed by the 
rectum. When floated in water they will be seen to in- 
volve the complete circumference of the intestine; they 
are generally gangrenous and emit a most offensive odor. 
Under the microscope the more or less altered structure 
of the part of the bowel from which they are derived may 
be demonstrated ; the examination will be made more easy 
if a drop or two of methylene blue be added to the water 
in which the specimens are teased out. 

Intestinal Concretions. — These objects are usually 
the result of accident, and are mere pathological curios- 
ities possessing no clinical value. Hard masses are often 
passed with the motions by lunatics ; thus Dr. Langdon 
Down records a case in which such a mass when examined 
proved to be cocoa-nut fibre. In other cases oat-hairs have 
been found matted together into a firm, solid ball. In rare 
instances, drugs which have been administered for too long 
a period, or incautiously, produce similar results, and 
masses of carbonate of magnesia or insoluble salts of iron 
may be found. 

• Mention may here be made of the occurrence of '* scy- 
bala" in the rectum, which often give much trouble, 
especially in elderly people. These are caused by the re- 



EXAMINATION OF THE F.ECES. 223 

tention of feces and removal of moisture, and the combi- 
nation of the constituents of the excrement with inspissated 
mucus. 

Gall-stones. — The passage of gall-stones is of great 
clinical interest, inasmuch as they often clear up a doubt- 
ful diagnosis. They are easily detected with the naked 
eye, and for closer examination may be obtained by shak- 
ing up the faeces with water, when they will immediately 
sink and are thus easily removed. 

Stones consisting of bile-pigment are the most common. 
They often occur in large numbers. They are black in 
color, irregular in shape, and sometimes present facets 
where they have rubbed against each other. They are 
friable and are easily crumbled. They vary much in size, 
but are usually about the size of hemp seeds. They are 
generally deposited round a nucleus, which consists of in- 
spissated mucus. 

Gall-stones are more rarely light in color, and then most 
probably composed of cholesterin. Their structure can 
easily be exhibited by scraping off a little with a knife 
and adding a drop of water. The characteristic crystals 
(see Fig. 43) are then seen, consisting of rectangular plates, 
with a notch out of one corner, superimposed one on 
another. Their nature may be further corroborated by 
adding to some of the scrapings a drop of sulphuric acid, 
when a brilliant red color will make its appearance. 
These calculi are smooth, and sometimes attain such a 
large size as to occupy the entire gall bladder. They are 
then, of course, only discovered after death. When several 
are present, they are faceted, and are much harder than 
those formed of bile-pigment. More rarely bile-stones are 
composed of carbonate or phosphate of lime. 

Before concluding the account of the macroscopic ex- 
amination of faeces, mention must be made of the bodies 



2 24 MEDICAL MICROSCOPY. 

described by Virchow and Nothnagel {Joe. cit., p. 96) 
resembling cooked sago grains or frog-spawn. Virchow 
thought that they came from an excess of starchy food. 

Finally the bodies known as ^' sable intestinal/' and 
described by Dr. Sheridan Delapine in the '' Pathologi- 
cal Transactions," xii, 1890, p. in, deserve brief notice. 

Labouline, in 1873, described small, irregular granules, 
not unlike cork-dust, which he had found in the stools, 
and gave to them the name ^^ sable intestinal." Dr. Del- 
apine has investigated several cases of this kind. 

The granules or small masses vary considerably in size, 
some being very minute, whilst others measure S/^ inch in 
their largest diameter. In one case they were brown in 
color, and were shown to be composed of vegetable cells, 
such as are found in sclerenchymatous tissue, a small 
amount of carbonate of lime, phosphates and oxalates, 
and a few silicious particles. In another instance they 
resembled coarse bran in appearance, and in yet another, 
small gall-stones. In the last case, however, it was shown 
that their nuclei consisted of undigested food matter, and 
that therefore they m.ust have been produced in the intes- 
tine, and also that they were not composed of cholesterin, 
or of any of the products which may accumulate within the 
gall bladder. After careful investigation Dr. Delapine 
came to the conclusion that all these masses were practi- 
cally nothing else than residues of articles of food (fruit 
pips, etc.) which had not been digested, either because they 
were undigestible, or because the stomach and intestine 
failed to digest them. Yet they could not be said to be 
simply undigested matters such as those found regularly in 
the faeces, since by their accumulation they gave rise to 
suspicious appearances and symptoms. 



examination of the f^ces. 225 

Microscopical Examination. 

The stools should be examined in two ways. First, by 
placing a small particle on a glass-slide, covering it with a 
cover-slip, and distributing it into a uniform layer by gentle 
pressure. Secondly, by shaking up a portion with many 
times its bulk of water, allowing the sediment to settle, 
then removing portions by means of a pipette, as in the 
examination of urinary deposits. 

In the stools in health various particles of undigested 
food, animal and vegetable, are met with, and the student 
should make himself well acquainted with these, although 
it is true that mistakes are not so liable to occur in the ex- 
amination of the faeces as in that of the sputum or urine. 
They are much the same as those which will be described 
as occurring in vomit (^. z/.). 

When the individual from whom the specimen is ob- 
tained is on a meat diet, muscular fibers will invari- 
ably be found, but are sometimes so altered as to be 
hardly recognizable, being swollen and stained yellow by 
the bile ; with a little care, however, their characteristic 
striation can usually be made out. With these fragments 
elastic fibers will also be seen ; they may be recognized 
by their clear outline, tendency to branch dichotomously, 
and the bold curves which are nearly always apparent. 
They are straighter and coarser than those described as 
occurring in human sputum, are very elastic, and are 
usually seen in masses. 

Bundles of areolar tissue will also be observed after 
meat has been taken, especially if the digestion is at all 
faulty; the fibers are irregularly arranged, and generally 
appear in a shapeless mass the exact structure of which is 
difficult to make out. Fat is usually found in the form of 
20 



2 26 MEDICAL MICROSCOPY. 

crystals, the needles being arranged in rosettes; these are 
especially numerous when the pancreas is diseased ; indeed, 
their presence in combination with almost colorless stools 
is strong corroborative evidence of blocking of the pan- 
creatic duct, but the exact nature of the lesion can, of 
course, only be made out clinically. 

Fat also occurs in globules. In chronic alcoholism it is 
found in abundance in the stools. 

When vegetables have been mixed with the diet, vege- 
table cells of various kinds are seen, spirals, hexagonal 
ceils, single or in groups, etc., the forms being too numer- 
ous to describe and depending upon the particular vege- 
table which has been eaten. 

Starch Granules. — Owing to imbibition these bodies 
are at first sight difficult to recognize, but their nature 
can easily be demonstrated by the addition to the 
specimen under the microscope of a drop of iodine in 
iodide of potassium (p. 41); the development of a deep 
purple color, turning to black when a drop of sulphuric 
acid is also added, proves the presence of starch. 

If only a milk diet has been given, as in infants, the 
appearance is different. The stools are loose instead 
of formed ; the constituents described above are absent, 
with the exception of fat globules and crystals, these being 
present in large numbers. Coagulated albumin (casein) 
is also found, usually as irregular yellow particles. 

Crystals. — In the microscopic examination of faeces 
several varieties of crystals will be met with, but very 
few of them have any diagnostic value. 

Fat Crystals (see above). — They occur very abun- 
dantly in the stools of patients suffering with jaundice. 
The investigations of Gerhardt, v. Jaksch, and Oesterlein 
show that they are formed of a combination of the alkaline 



EXAMINATION OF THE F.ECES. 227 

earths with the higher fatty acids. The crystals take the 
form of minute, sharp-pointed crystals, scattered through the 
field or arranged in clusters like rosettes. 

Hsematoidin Crystals. — The author has never found 
these crystals in the alvine discharges, but v. Jaksch states 
(/. ^., p. 156) that he has not infrequently done so, espe- 
cially in chronic intestinal catarrh from over-eating, and 
in many instances in which blood had been discharged 
into the intestine some time before the stools were passed. 
The form of the crystals is usually ill defined ; they are 
sometimes free and sometimes enclosed in masses of a 
shining substance resembling mucin. 

Charcot-Leyden Crystals (for an illustration of 
these bodies see Fig. 54). — As these crystals are the 
result of decomposition of albuminous substances, it is 
only to be expected that they are sometimes found in the 
stools. They have, however, no diagnostic significance, 
and have been found in various diseases. 

Oxalate of Lime Crystals (Fig. 37, p. 203.) are fre- 
quently met with and are probably derived from the food, 
being particularly abundant after a vegetable diet. Other 
crystals occasionally found, but having no clinical value, 
are : triple phosphate, phosphate of lime, carbonate of 
lime, and sulphate of calcium. 

The formed elements which are found in the motions 
are blood corpuscles, white and red, and epithelial cells, 
but the most important, from a practical point of view, are 
the various parasites with their ova, and some pathogenic 
micro-organisms. The same plan will be adopted in their 
description as in the chapters on the examination of 
sputum, urine, vomit, etc. 

Red Blood Corpuscles. — The occurrence of blood in 
the stools has already been briefly alluded to (p. 219), but 
although the coloring matter (somewhat altered by the 



2 28 MEDICAL MICROSCOPY. 

action of sulphuretted hydrogen) is frequently present, 
yet the red blood cells are very seldom seen, even when 
the blood is apparently abundant, as in typhoid fever. 
Small, irregular particles of haematoidin, reddish-brown in 
color, are met with, and more rarely, crystals of this sub- 
stance. The presence of blood may be demonstrated 
thus: A small portion of the excrement is dried, and the 
powder placed on a glass slide ; a crystal of common salt 
is then added, together with a few drops of glacial acetic 
acid. The specimen is covered with a cover-glass, and 
heated until steam just begins to rise ; after cooling, 
minute crystals of haematin will be visible by aid of the 
microscope. 

Leucocytes. — White blood cells are but seldom met 
with, even when there is considerable inflammation of the 
intestine. If ulceration takes place, pus cells may be found 
in considerable quantity. The discharge of an abscess into 
the bowel is indicated by the appearance of pure pus. 

Epithelium. — Epithelial cells in greater or less abun- 
dance are always found in the faeces, and their presence, 
therefore, is of very little clinical importance. 

Two chief varieties are met with, squamous cells from 
the lower end of the rectum, and cells which are fusiform 
or columnar in shape, from the remainder of the intestinal 
canal. These latter are sometimes difficult to define, their 
outlines being faint, but their nuclei are always seen, and 
are especially distinct if some dilute staining fluid be added, 
such as methylene blue or magenta. 

Nothnagel has drawn especial attention to the fusiform 
cells, which he considers a degenerative form, caused by 
the abstraction of fluid. 

In addition to the above constituents of the evacuations, 
a certain amount of amorphous material is always present, 
derived chiefly from the waste products of digestion ; it is 



EXAMINATION OF THE FAECES. 229 

of little importance medically, either with a microscopic or 
on chemical examination. 

The subject of parasites discharged from the bowel is 
a very large one. It will be most convenient to follow the 
usual division into vegetable and animal parasites, subdi- 
viding them into those which are pathogenic and those 
which are not. The former, being the most important, will 
be considered first in each division. 

Vegetable Parasites. 

Pathogenic. 

Bacillus of Tubercle. — Tubercle bacilli are fre- 
quently found in the stools of phthisical patients afflicted 
with diarrhoea, and in such cases ulceration of the intes- 
tines is almost invariably found after death. It is, of 
course, conceivable that the bacilli may be derived from 
the sputum which has been swallowed, but if such were the 
case, they would be very few in number, and it would be 
no proof that an intestinal lesion did not also exist, as 
ulcers are frequently found after death, in which diarrhoea 
was not a prominent symptom during life. 

It is not, however, with obviously consumptive patients 
that an examination of the faeces for tubercle bacilli is of 
value, but in those cases in which there is obstinate diar- 
rhoea, with raised temperature, and in which the diagnosis 
is doubtful. If the organisms are then found the uncer- 
tainty is cleared up, and if they are very plentiful, so as to 
resemble almost pure cultures, ulceration is nearly certain to 
be present. In order to search for tubercle bacilli, a small 
portion of the faeces is placed between cover-glasses, which 
are dried, passed three times through the flame, and stained 
in the same way as will be described in the chapter dealing 
with the examination of sputum. 



230 MEDICAL MICROSCOPY. 

Bacillus of Typhoid Fever. — The pathogenic value 
of the bacillus first described by Eberth in 1880 in causal 
relationship with enteric fever is now accepted by most 
bacteriologists. The bacilli, however, unlike those of 
tubercle, possess no characteristic staining property ; it is, 
therefore, impossible to demonstrate them satisfactorily in 
typhoid stools, although they may readily be demonstrated 
in the tissues after death by the process described on p. 97 
(Loffler's method). 

To prove their presence in the faeces a troublesome and 
prolonged process of separation of the colonies by plate 
cultivations has to be adopted, and this can only be ac- 
complished in a properly appointed bacteriological labo- 
ratory. 

Bacillus of Cholera. — The discovery by Koch of the 
** comma bacillus," which has now been conclusively 
proved to be the cause of Asiatic cholera, was the chief 
means of placing bacteriology in the position which it 
now holds in modern science. Fortunately, in England at 
the present day we have very rare opportunities of studying 
the cholera bacillus, except in laboratories. Koch found 
the organism in the intestine and alvine discharges of 
patients who had died or were suffering from Asiatic 
cholera. It was rarely found in the vomit, and never in 
the other secretions, urine, saliva, etc., nor in the blood or 
breath. 

The bacillus is a short, thick rod, not so long as the 
tubercle bacillus, and generally more or less curved. The 
organisms are often united together into wavy threads 
(spirilla), especially in artificial cultures, owing to the 
rapidity of reproduction. As with the bacillus of enteric 
fever, the cholera organism is very difficult to demonstrate 
in the stools, owing to the myriads of other bacilli which 



EXAMINATION OF THE FiECES. 23 1 

are present, but as in the typical rice-water stools it often 
occurs in huge numbers, an attempt may be made to do so 
in the following manner : — 

A small portion of the flaky sediment of the stools is 
placed on a cover-glass, which is then covered by a 
second, so as to obtain two uniform layers by gently 
sliding the glasses apart. They are dried and the films 
fixed by passing them three times through a flame. They 
are then placed, prepared side downward, in a watch-glass 
containing an alcoholic solution of fuchsine or methylene 
blue. After five minutes they are removed and thoroughly 
washed in water, after which they are dried and mounted in 
balsam. 

More satisfactory results are obtained by adopting Koch's 
method of plate cultivation, but this requires considerable 
experience in bacteriology. It may, however, be briefly 
stated here that, cultivated on plates of nutrient gelatine, 
the colonies commence to form in about twenty-four hours ; 
they are white in color, roughly circular in shape, but 
have irregular outlines. Gradually a yellowish tint ap- 
pears, which grows darker, especially toward the centre, 
and the gelatine begins to liquefy, emitting a most un- 
pleasant odor. 

For further descriptions of this bacillus and its growth 
in other media, the reader is referred to the text-books on 
bacteriology. 

Bacillus of Cholera nostras. — This organism is 
often known as the ^' bacillus of Finkler and Prior," these 
observers having first described it. It is found in the 
stools of patients afflicted with '* cholera nostras," and 
may be demonstrated in the same way as the bacillus of 
Asiatic cholera; it differs from the last named in being 
broader and larger, but the most marked differences are 
seen in the cultures on nutrient gelatine. The colonies 



232 MEDICAL MICROSCOPY. 

are very much larger than those of the comma bacillus of 
the same age. They have a faint yellowish-brown tinge, 
the borders are well-defined, and they exhibit a distinctly 
granular appearance. They liquefy nutrient gelatine very 
rapidly, so that the first plate of a series may be com- 
pletely liquefied on the day following inoculation, emitting 
a very foul odor. 

Non-Pathogenic Organisms. 

Micro-organisms of various kinds abound in the faeces, 
both in health and disease. So numerous are they that 
they often form a considerable proportion of the whole 
bulk of the stools. It is on account of this that pathogenic 
organisms such as those of typhoid or dysentery are almost 
impossible to recognize, not possessing any characteristic 
selective powers for particular stains. 

All the fission fungi stain readily with the ordinary 
stains, fuchsine and methylene blue being the best. A 
small portion of the discharge is spread out into a uni- 
form layer between cover-glasses, which are then separated 
by a sliding movement, as in the examination of sputum, 
dried, and passed three times through the flame. The 
glasses are then placed face downward in a watch-glass 
containing the undiluted stain, or a drop or two of the 
dye is placed on the glasses. After three minutes, the 
preparations are washed thoroughly in water j if fuchsine 
be used, a few drops of methylated spirits may be added. 

Very excellent specimens may be obtained by means of 
the Gram method. After the glasses are prepared in the 
ordinary way, a few drops of methyl-anihne-violet are 
placed on them and allowed to remain for three minutes, 
they are then washed in Gram's solution (p. 41) for one 
minute, and are finally dipped alternately in aniline oil and 
methylated spirits until no more color comes out, after 



EXAMINATION OF THE F.ECES. 233 

which they are dried and mounted in balsam. Another 
method by which good results are obtained, and which, 
as will be presently again mentioned, is strongly recom- 
mended by V. Jaksch, is to immerse the glasses for about a 
minute into a solution of iodine in iodide of potassium. 
This reagent has the advantage of staining the yeast fungi 
as well as bacilli and micrococci, and for some of the latter 
it has a peculiar power of differentiation, coloring them 
blue or violet. 

Moulds. —Members of this group are very seldom 
found in the faeces when first passed. Practically the 
only one ever met with is the thrush fungus — Oidium albi- 
cans, which consists of small round spores and mycelial 
threads. It is generally found in the stools of children 
suffering from a similar condition in the mouth. It is of 
very slight diagnostic importance. . 

Saccharomycetes. — These are very commonly found 
in the alvine discharges, both in health and disease, occur- 
ring as round or oval cells, exhibiting the characteristic 
gemmation ; they are especially abundant in the acid stools 
of children. They are also found in large numbers in 
acute catarrh of the small intestine in adults. When a 
cover-glass preparation is stained with iodine solution, the 
cells assume a mahogany-brown color. 

Schyzomycetes. — The fission fungi often occur in 
such large numbers as to constitute the main bulk of the 
discharge. In sprue, for instance, the peculiar whitish 
scum found in large quantities in the stools in the exacerba- 
tions of the disease, consists entirely of bacilli of various 
sizes. Dr. Thin has isolated thirteen varieties j probably 
one of these is pathogenic, but neither Dr. Thin nor the 
author has been able to verify this assumption. 

The organism most frequently met with is the one asso- 



234 MEDICAL MICROSCOPY. 

ciated with ordinary putrefaction, namely, the Bacterium 
termo. 

Nothnagel was the first to describe the Bacillus subtilis 
in the stools. It has no pathological value. It occurs as 
long mycelial threads, the spores being situated at the end 
of the rods or attached to the threads. These micro- 
organisms stain brownish-yellow with the iodine solution. 

Von Jaksch, in a most exhaustive investigation on this 
subject, has described a large number of fungi which stain 
blue with the iodine solution, none of which, however, 
have any clinical value (^^ Clinical Diagnosis," p. 132). 
Some of them are micrococci, which occur in large num- 
bers, and are colored reddish-violet with the solution. 

Next in order are small bacilli resembling those of mouse 
septicaemia ; these stain in a similar manner. 

Larger rods are sometimes seen, which resemble the 
Leptothrix buccalis. Finally, large round cells are met 
with, strung together like beads, appearing very much like 
yeast cells. 

It may be here remarked that the coloring produced by 
the iodine solution is only transitory and has usually com- 
pletely disappeared in forty-eight hours. 

Animal Parasites. 
Protozoa. — Various forms of monads and infusoria are 

met with in the faeces, but are comparatively uncommon, 
and merit only passing notice. 

Nothnagel found monads in the stools of consumptive 
and typhoid patients ; active movements had generally 
ceased. 

The Amoeba coli was first described by Losch (^^ Vir- 
chow's Archiv," 1875, P- ^9^)- These bodies are circu- 
lar in form, contractile, and consist of coarsely granular 
protoplasm, and possess a round nucleus. 



EXAMINATION OF THE Fi^CES. 235 

Some valuable researches on amoebic dysentery have 
recently been made by Drs. Councilman and Lafleur 
('* Johns Hopkins Hospital Reports," December, 1891, 
vol. ii, p. 395, et seq.). They found in certain cases of 
dysentery amoebae in the stools which did not differ materi- 
ally from those described by Losch. In searching for 
the organisms some choice had to be exercised in the por- 
tions of stools to be examined. They were more numer- 
ous in the small gelatinous masses which were often .con- 
tained in the faeces than elsewhere. The numbers which 
were found in different cases, and even in the same case at 
different times, varied greatly. At times no single portion 
of the faeces which could be examined was free from them. 
In other cases single individuals were only found after long 
and careful search. Their numbers in general were pro- 
portional to the severity of the lesions. 

Drs. Councilman and Lafleur state that the amoebae differ 
somewhat in appearance according as they are active or 
inactive. In the latter condition they are round or slightly 
oblong, and are more highly refractive than the other cells 
which may be found in the stools. In this resting condi- 
tion there can frequently be distinguished no division into 
an ecto- and an endo-sarc, but there is simply a body, en- 
closing vacuoles of greater or less size. They are difficult 
to recognize in this condition unless active ones are found 
at the same time and so attention be directed to their pres- 
ence, but when in motion their appearance is characteris- 
tic. Every degree of activity of movement can be seen. 
At times the change is so slow that one must look carefully 
for some time to be sure that any motion is taking place, 
and at others the movements are so rapid that certain of 
their phases can scarcely be followed. The movements 
consist essentially of two kinds : one progressive and the 
other limited to the thrusting out and retraction of pseudo- 



236 MEDICAL MICROSCOPY. 

podia. In some instances there seems to be a certain 
rhythm in these changes of form and position, which occur 
at regular intervals. 

Various bodies were frequently enclosed in the amoebae. 
The most common of these were red blood corpuscles, 
often in considerable numbers. Pus cells, well preserved, 
or in various stages of disorganization, were also seen. 
Both bacilli and micrococci were occasionally enclosed. 
In some cases, in addition to red blood cells, blood pig- 
ment was found, either in the form of black granules or 
as irregular brown masses. 

Of infusoria the most common is the Cercomonas 
intestinalis. It occurs in the mucous discharges of 
young children. It is pear-like in shape, furnished with 
tentacles, and has a clear nucleus. 

An organism closely resembling the one first described 
is the Trichomonas intestinalis, but distinguished 
from the former by a ciliated disk at one end. 

Another entozoon occasionally found, especially in 
diarrhoea, is the Paramsecium coll. It is oval in shape 
and covered with cilia, and the anterior extremity is nar- 
rower than the posterior. 

Vermes. 

The recognition of the various worms which infect the 
human intestinal canal is of great importance, not only 
with a view to undertake treatment which may expel them, 
but also as a means of clearing up vague symptoms which 
have previously evaded explanation. 

It is not intended here to enter into the full life history 
of each parasite, and to trace it from the ovum through the 
'* intermediate host" to the human intestine; but only 
those forms will be described which are likely to come 
under the notice of the medical man in his clinical work. 



EXAMINATION OF THE F^CES. 237 

In addition to the worm, the ova are sometimes found 
in the stools ; these are never discharged through the 
genital canals of the parasite, but remain in situ until the 
proglottis is ruptured. 

Cestoda. 

The tape-worm as a whole is known as a ^^ strobilus/^ 
and is divided into a ** head/' '^ neck," and a number of 
segments or proglottides. 

Tsenia mediocanellata (Saginata). — This is the most 
common tape-worm in this country. The intermediate 
host is the ox. 

The head, which is the most easily recognized part of 
the parasite (Fig. 44), is square in shape, and measures in 
the transverse diameter 1.5 mm. It is provided with four 
suckers, which usually have much pigment deposited round 
them. 

As the head is generally difficult to find, or may not 
come away when the rest of the strobilus is discharged, it 
is important to be able to recognize the proglottides. The 
whole worm may attain the length of four yards, and 
acquires its full growth in about three months, so that if 
the head has not been discovered, although proglottides 
have been passed, we may conclude that the parasite has 
been destroyed, if no fresh segments are discharged for 
thirteen weeks. There is no distinct neck, but the first 
segment appears immediately below the head. The upper 
segments are broad and short; about the centre of the 
animal they become square, and in the lower half the 
length exceeds the breadth. 

The sexual organs attain their full development about 
the 450th joint from the head, the ova appearing about 
400 joints lower down. 

The ripe proglottides measure ^ inch in length and 



238 MEDICAL MICROSCOPY. 

about Ys inch in breadth. They usually rupture and dis- 
charge their ova while in the intestine, so that those which 
are passed per anum are shriveled and empty (Fagge). 

The segments may be recognized and distinguished 
from those of the other tape-worms by the form of the 
uterus. This organ has numerous branches, having twenty 
or thirty on each side of a median channel; the branching 








Head of Taenia mediocanellata (Boyce). 

is dichotomous, and the terminations are round and club- 
shaped. The genital pore does not alternate regularly, 
but may be situated on the same side in two or three suc- 
cessive segments ; they are. however, always situated on 
the narrow edge. 

Malformations are particularly liable to occur, and 
sometimes there are two or three genital pores in a single 
proglottis, each corresponding with a separate double 



EXAMINATION OF THE Fi^CES. 239 

sexual apparatus; sometimes the segments are not regularly 
developed, so that imperfect triangular joints disturb the 
symmetry of the series ; or a supernumerary proglottis 
projects by the side of the continuous hne of joints ; very 
rarely there are two distinct chains united in their whole 
length by one edge at an acute angle. 

The eggs are oval, and exhibit the primordial yolk 
membrane ; no booklets are seen in the embryo, as is the 
case in Taenia solium. 

Taenia solium. — This worm was so named because, 
according to an old idea, it was thought to exist alone in 
the intestine. This is now known not to be the case ; 
sometimes they occur in large numbers, as many as five- 
and-twenty having been passed by one individual. It was 
also formerly supposed to be the most common of the tape- 
worms in this country, but this, too, has been shown to be 
without foundation, the Taenia mediocanellata occurring far 
more frequently. 

The head of the Taenia solium (Fig. 45) is of a generally 
rounded form, about the size of a pin's head. In front it 
is prolonged so as to form a proboscis or rostellum, which 
is surrounded by a circle of twenty-six booklets arranged 
with their points outward. They are of two sizes, large 
and small alternately. The wide part of the head is pro- 
vided with four large sucking discs, and is generally black 
from the presence of pigment. Owing to its small size, it 
is difficult to find, and is frequently overlooked by the 
patient, who should be directed by the physician to bring 
for examination every particle of white matter passed. 

The loss of the head is rendered more probable by the 
very slender neck which it surmounts. This neck is about 
J^ inch in length, and it gradually merges in the anterior 
part of the body, in which fine transverse lines begin to 
appear at the first indication of the formation of segments. 



240 MEDICAL MICROSCOPY. 

On passing down, the segments elongate and become 
more completely divided. The whole length of the 
strobilus is usually about nine feet. The joints are at first 
very small, and broader than they are long. They gradu- 
ally increase in breadth and length, until about the middle 
they appear square, and in the lower half they are longer 
than they are broad. 

Fig. 45. 




/ 




Head of Taenia solium (Boyce). 

The proglottides may be distinguished from those of the 
mediocanellata by the shape of the uterus. This is more 
simple in the solium, consisting of a central passage 
running parallel to the length of the proglottis, and 
having about ten branches on each side, which ramify 
instead of dividing dichotomously. The genital pore is 
placed in a little papilla, situated on the lateral edge of the 
segment. It alternates regularly on the two sides of the 
animal throughout its whole length. 



EXAMINATION OF THE FAECES. 24I 

Proglottides are best demonstrated by placing two of 
them on a glass slide and covering them with a large 
cover-glass, when they may be examined with a good lens. 
The specimens will be rendered more transparent by the 
addition of a little glycerine. 

The total number of segments is about 800. The sexual 
apparatus begins to appear about the 200th segment from 
the front, and is mature about the 450th. Its time of 
growth is the same as for the mediocanellata, and the same 
deductions may be drawn from the appearance of the seg- 
ments. 

The ova (Fig. 46, g) are globular, and they measure .036 
mm. in their longest diameter. They have a shell which 
appears to be marked with a number of minute radiating 
lines, this being due to rod-shaped projections, which 
closely cover its surface ; when mature, embryos fur- 
nished with booklets are seen within the eggs. The 
intermediate host of this parasite is the hog, hence its 
greater frequency on the Continent, where uncooked pork 
is a frequent article of diet. 

Taenia nana is the name given to a form of tape-worm 
prevalent in Sicily, Italy, and Egypt. Ranson mentions 
it as having occasionally been found in England. Its 
average length is nearly j4 a.n inch, and its breadth about 
Jq- inch. It has a circular head provided with four suckers, 
and a rostellum surrounded by about twenty booklets. 
The uterus is oblong. This parasite occurs in huge num- 
bers in a single patient. 

Bothriocephalus latus. — This worm is limited to the 
inhabitants of certain countries of Europe, being most 
commonly found in Switzerland and Sweden. It is the 
largest of all the tapeworms, attaining a length of from 16 
to 20 feet, and possessing from 3000 to 4000 segments, 
which are broader than they are long. In the middle of 
21 



242 MEDICAL MICROSCOPY. 

the strobilus they are about y^ an inch broad by \ inch in 
length, but the last ones gradually become longer and 
narrower, so as to be almost square. This worm has 
a longitudinal projecting ridge traversing its whole 
length. The uterus occupies the middle of each segment. 
It is composed of a convoluted tube bent several times 
upon itself, so as to give a rosette-like appearance. The 
genital pore lies in the centre of each proglottis, opening 
upon its ventral surface. When portions of the worm are 
passed by a patient, the segments do not come away 
separately, but portions two and three feet in length are 
expelled. The head of this parasite is oval and about -2^^ 
inch in breadth. It is blunt at the anterior extremity and 
has two deeply grooved longitudinal suckers, one on each 
side. 

The eggs (Fig. 46, d) are oval, and are about .07 mm. 
in length. They are covered with a brown shell, and open 
in a peculiar manner at one extremity by a kind of small 
lid, so allowing the embryo to escape. 

The intermediate host of this tapeworm is not known, 
but is supposed to be some fish. The frequency of fresh- 
water lakes in Switzerland may explain its prevalence in 
that country. 

Other tapeworms which are occasionally found in the 
human subject, but are so rare as only to need mention 
here, are the Tcenia cucit7nerina, which is about 8 inches 
long, having a small head witha rostellum surrounded with 
a quadruple circle of booklets j the Tcenia flavopuncta ; 
the Tcenia madagascariensis, and the Tania lepiocephala. 

The ova of the above Cestodes are occasionally found 
in the faeces without any segments accompanying them. 
Should the presence of a tapeworm be suspected, the stools 
should be mixed with water and the sediment allowed to 
subside, and this process should be repeated several times 



EXAMINATION OF THE FAECES. 243 

until very little matter remains ; a small portion of this 
should be placed on a glass slide, a cover-glass applied, 
and the specimen examined by a ^ inch lens, when the 
eggs, if present, will be easily recognized. 

The same process should be adopted in searching for the 
smaller parasites now to be described. 

Trematoda. 

These worms are commonly known as ^^ Flukes," they 
are flat and more or less oval in form. On their ventral 
surfaces they have one or more sucking discs. They are 
rare in man, but have occasionally been found in the 
stools, or more often in the biliary passages or intestines. 

Distoma hepaticum, — This worm is very common 
amongst sheep, producing the disease called '^Rot." As 
its name implies its chief habitat is the liver. It frequently 
dilates and obstructs the bile ducts. It is about an inch 
in length and half an inch in width. The body is flat 
and is elongated anteriorly, forming a kind of a head 
which is furnished with a sucker. There is another sucker 
on the ventral aspect. Between the two lies the genital 
pore leading to the uterus, which is convoluted. The eggs 
(Fig. 46, e) are small oval bodies, .13 mm. long; they are 
brown in color, and one end, which is broader than the 
other, opens by a small lid. 

Distoma lanceolatum. — This species is about a ^ inch 
long and yig- inch in breadth. It is pointed at the extremi- 
ties, but more so anteriorly than posteriorly. It has no 
alimentary canal, but possesses a complicated generative 
apparatus packed with numerous ova. These are oval, 
brown in color, and about .04 mm. in length. This tre- 
matode is very rarely seen in man, and only occurs in 
comparatively small numbers in cattle. 

Distoma sinense. — This parasite is more elongated 



244 



MEDICAL MICROSCOPY. 



than the two just described. It is about J^ inch in length 
and about ^ inch in breadth. It possesses a single suc- 



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EXAMINATION OF THE FAECES. 245 

end. When seen in the bile in the fresh state, a beautifully 
delicate green color, tinged with yellow, may be observed 
round the edges, whilst the centre of the animal is a deep 
brown. The ova are extremely small and occur in very 
large numbers. This worm only occurs in man, and as 
many as five hundred may be found in the bile ducts of 
one person. Hitherto it has only been described among 
the Chinese. 

Nematoda (Round Worms). 

Ascaris lumbricoides (common round worm). — 
The Ascaris lumbricoides inhabits the small intestine, but 
may be passed either alone, or with the stools. In general 
appearance it closely resembles an ordinary earthworm. 
Whilst living it has a reddish-brown color, fading after 
death to a duller gray. It measures six to sixteen inches 
in length, is marked by transverse striae, and tapers toward 
both extremities. The female is about twice as long as the 
male ; the head consists of three conical projections which 
are furnished with fine teeth and touch corpuscles. The 
posterior extremity of the male is folded on the anterior 
surface like a hook. The ova (Fig. 46,/") are circular or 
bluntly oval ; yellowish-brown in color, and measure .07 
mm. in length. They are covered with an albuminous 
sheath which is often tinged with bile. Beneath this 
covering is a dense shell. Several of these parasites (three 
to four hundred) may be passed by one individual ; but 
probably each worm only exists for a few months within 
the body of its host. 

Ascaris mistax. — This parasite in general form much 
resembles the preceding, but is smaller, and the shape of 
its head is rather more pointed. Hitherto it has only 
been found in the cat. 

Oxyuris vermicularis (threadworm). — As their 



246 MEDICAL MICROSCOPY. 

common name indicates, these worms are like small ends of 
white thread ; the male being about an eighth of an inch 
and the female about four-tenths of an inch in length. 
They have a comparatively blunt anterior and a tapering 
caudal extremity. The tail of the male is provided with 
six pairs of papillae. The male is much more frequently 
seen than the female. 

The eggs (Fig. 46, b) are oval in form and flattened on 
one side. They measure .05 mm. in length. The con- 
tents are granular except when mature, when the embryo, 
may be seen. The shell is membranous and presents a 
treble contour. 

Trichocephalus dispar (whipworm), Fig. 47). — 

Fig. 47. 




Trichocephalus dispar. 

This parasite inhabits the caecum. It is cylindrical in 
form, having a short thick body, and long whip-like neck. 
The male is about i}^ inches in length, and the female 
about 2 inches. The worm fixes its thin extremity by 
means of four claw-like teeth into the wall of the intestine 
and is therefore only very rarely found in the faeces. The 
ova (Fig. 46, a) are oval and are about .05 mm. long, and 
are characterized by having a translucent point at either 
extremity. 

Anchylostoma duodenale (Strongylus duodenalis). 
— This parasite does not occur in this country, but is fre- 
quently met with in Egypt and Brazil. It inhabits chiefly 



EXAMINATION OF THE FAECES. 247 

the jejunum. It is cylindrical in form, the male measur- 
ing about half an inch, and the female an inch in length. 
The head, which is bent nearly at right angles to the body, 
is armed with four conical teeth, by means of which the 
animal fixes itself on the mucous membrane. The pos- 
terior extremity is tapering, and in the male extends into 
a pouch with three flaps. The eggs (Fig. 46, c) are oval, 
with a thin, transparent shell, and have a long diameter of 
.05 mm. The division of the yolk (Fig. 46, c') can be 
plainly seen. This nematode is only seen in the stools 
after some measures have been taken to expel it, but the 
eggs frequently appear. 

Strongylus gigas. — This is a large worm chiefly met 
with in the kidney, bladder, lungs, and liver of dogs, but 
a few cases are on record in which it has been met with in 
the pelvis of the kidney in man. It is a large worm, the 
male being about ten inches in length and the female nearly 
a yard. 

Anguillula intestinalis. — This is a small, round 
worm, which has been discovered in the stools in cases of 
Cochin-China diarrhoea, but has not been met with in this 
country. 

Trichina spiralis. — These parasites have in very rare 
cases been found in the stools. They closely resemble the 
Trichina found in muscle, but are not encapsuled. The 
male is 1.5 mm. in length and the female 3 mm. The 
males are distinguished from the females by haying two 
conical projections from the posterior extremity. 



CHAPTER XVI. 

EXAMINATION OF SPUTUM. 

By '^ sputum" is understood all the matter coughed or 
hawked up from the air passages. As this material includes 
the secretions from the buccal respiratory tract, it might 
naturally be expected that an examination of the contents 
of the spittoon would yield valuable results as regards the 
condition of the parts forming that tract. Since the dis- 
covery of the tubercle bacillus by Koch, in 1882, the mi- 
croscopic examination of sputum has received a great im- 
petus, and it has now become almost a matter of routine 
in any case of lung disease of a doubtful character. 

The naked eye features of the expectoration received 
much more attention from the older writers, before the 
physical examination of the chest by percussion and auscul- 
tation arrived at the present state of exactness, and before 
the clinical thermometer had been brought into routine use. 
Not only were the appearances of the secretion noticed, 
but attention was directed to its smell, taste, reaction, etc. 
With our modern methods of physical examination, much 
less stress is now laid on the macroscopic characters of 
sputum, but with the aid of the microscope a diagnosis, 
which may be doubtful or extremely obscure (<f.^., Acti- 
nomycosis), may be corroborated or established. 

Although this chapter will necessarily be directed chiefly 
to the microscopic examination of sputum, it will be 
necessary, or at any rate desirable, to briefly consider 
some of the more noticeable points in connection with its 
quantity, color, odor, etc. 

248 



EXAMINATION OF SPUTUM. 249 

Sputum may be described as mucous, muco-purulent, 
or purulent, according as it is transparent, semi-opaque, or 
resembles the matter drawn from an abscess. To attempt 
to describe its examination under these heads would be 
manifestly absurd, as instances of each are found in the 
course of most diseases of the respiratory organs. 

It is very important that a satisfactory sample should be 
obtained. In the first place, then, the spittoon must be 
carefully washed out before use, and must never be made a 
receptacle for grape skins and various other odds and 
ends which seem so frequently to find their way into it. 
It is well also to replace it with another during meal times, 
otherwise particles of food are certain to become mixed 
with the expectoration, which gives much unnecessary 
trouble to the physician when searching for the minute 
white particles so characteristic of certain diseases. This 
is especially the case if the patient be on a milk or farina- 
ceous diet. Thus, in the sputum of one case which I had 
sent me for examination in several succeeding samples, min- 
ute white specks were seen, which, combined with the his- 
tory and symptoms of the case, had given rise to a suspicion 
that it was one of actinomycosis. By means of micro- 
chemical tests, however, they were shown to consist simply 
of starch grains derived from food. 

Sputum coughed up early in the morning before the 
patient has partaken of any food is to be preferred, though 
for some purposes the whole quantity collected during 
twenty-four hours is necessary. 

In order to observe the stratification peculiar to some 
diseases, the sputum should be collected in conical glasses. 

Quantity. 
The amount of sputum expectorated within twenty-four 
hours varies very greatly. In some conditions the spittoon 
22 



250 MEDICAL MICROSCOPY. 

may be filled several times daily, whilst in others there will 
scarcely be a drachm. The sputum is excessive in oedema 
of the lungs and in some cases of haemoptysis ; also when 
an abscess bursts, derived either from the lung itself or 
breaking through into the lungs from neighboring parts. 
Most commonly this is due to the rupture of an empyema, 
but the pus may be derived from the abdominal cavity. 
The quantity is also excessive in bronchiectasis ; the patient 
may rest for several hours, suddenly bringing up many 
ounces of fetid purulent matter. 

In phthisis again, where large cavities exist, the expec- 
toration is often very copious, but this is by no means 
characteristic. 

Consistence. 

The consistence of the sputum stands usually in some 
relation to its quantity, for if this be very copious the 
expectoration is generally thin and watery, whilst if small 
in amount it is usually thicker j but numerous exceptions 
to this statement are constantly met with. The consistence 
is dependent in a great measure upon the amount of mucoid 
material contained in it. As a type of a tough, tenacious 
sputum, that occurring in croupous pneumonia may be 
taken ; it is often so tenacious that if the spittoon be 
turned upside down, it will not flow out. The reason for 
this is difficult to assign, for the proportion of water is not 
small, nor is the quantity of mucin contained nor its 
specific gravity unusually large. The expectoration in 
bronchial asthma is often very tough, as can be proved by 
the difficulty experienced in obtaining a thin layer for 
microscopic examination ; it contains a large amount of 
mucin. In the early stages of acute bronchitis also the 
secretion is very thick and tenacious. This condition was 
termed by the older writers Sputum crudum, in contradis- 



EXAMINATION OF SPUTUM. 25 1 

tinction to the thin, watery expectoration, which they 
named Sputum coctum. 

Color. 

In speaking of the color of sputum we must consider 
briefly its transparency. Pure mucoid expectoration is 
colorless and almost transparent. The more cellular ele- 
ments there are present, especially leucocytes, the more 
cloudy and opaque the secretion becomes. 

Changes in color are almost entirely due to the presence 
of blood or its derivatives. 

This is not the place to enter into a discussion on the 
different causes of haemoptysis. Roughly speaking, they 
may be divided into two classes, in the first of which a 
rupture of a blood vessel, either in the pulmonary or 
systemic circulation, gives rise to the hemorrhage, whilst 
in the other there is a diapedesis of red blood corpuscles — 
parenchymatous hemorrhage. The amount of blood may 
vary from fine streaks distributed through the expectora- 
tion to almost pure blood. The latter condition is only 
found when a vessel has completely ruptured. Investiga- 
tions by Dr. Kidd and Mr. Taylor at the Brompton 
Hospital for Consumption showed that in a large propor- 
tion of the cases of profuse haemoptysis, a small aneurism 
had burst, whilst erosion of the vessels was comparatively 
rare. When first ejected the blood is usually bright in 
color, frothy in appearance, and alkaline in reaction. 

Haemoptysis is by no means diagnostic of tubercle ; 
heart disease, especially mitral stenosis, bronchitis and 
pneumonia, croupous and catarrhal, congestion of the 
lungs, infarcts and the presence of parasites, new growths, 
etc., all may give rise to blood spitting more or less pro- 
fuse. Professor iVlbert Frankel in a most exhaustive 
discussion ('* Pathologie und Therapie der Krankheiten 



252 MEDICAL MICROSCOPY. 

des Respirations Apparates," vol. i, p. no) on the occur- 
rence of haemoptysis, cornes to the conclusion that blood 
spitting without any previous symptoms is most probably 
always the expression of commencing tubercular inflam- 
mation. 

If the blood has been retained in the lungs for any time 
it undergoes changes which produce considerable alteration 
in its color and general appearance, this being due to 
decomposition or metamorphosis of the haemoglobin. 
Thus in gangrene of the lungs, the expectoration becomes 
of a dirty reddish-brown color, owing to the formation 
of methaemoglobin and haematin. 

Very characteristic also is the peculiar chocolate color 
which the expectoration assumes when a croupous pneumo- 
nia is passing into gangrene. This form was termed by 
Andral '^ prune-juice expectoration." Under the micro- 
scope the blood corpuscles will be found to have undergone 
very considerable changes, which will again be referred to 
later on. The same appearance is sometimes noticed in 
the sputum of aged persons suffering from oedema of the 
lungs. A peculiar brownish color of the expectoration 
is often found in gangrene of the lung, more commonly 
when ulceration of the lungs has occurred from some other 
cause. Leyden considered this to be due to the abundance 
of hsematoidin and bilirubin crystals which are present. 

Brown induration of the lungs produces a peculiar ap- 
pearance in the sputum. In the mass of mucoid material 
rust-colored, streaky deposits are seen, especially in the 
early morning, and these under the microscope are found 
to consist of pigmented epithelial cells. 

More common than the above is the well-known rust- 
colored sputum of croupous pneumonia. This color, 
however, is not always constant ; occasionally it assumes a 
lemon-colored hue, or more commonly a grass-green 



EXAMINATION OF SPUTUM. 253 

color, this being especially the case when the crisis has 
been deferred. This latter appearance must not be con- 
fused with those serious cases in which jaundice has super- 
vened, for the green color is then due to admixture with 
bile-coloring matters. 

Traube also described this green color in subacute cases 
of pneumonia, so that in any doubtful case when green 
expectoration is observed, micro-organisms should always 
be sought for. 

Various observers have described a yellow-ochre - 
colored sputum, this being associated with abscess of the 
liver, more especially when hydatid cysts are present. 
Under these circumstances large numbers of haematoidin 
and bilirubin crystals are seen under the microscope and 
the booklets of the parasite may be discovered on micro- 
scopic examination. 

The above colorations must not be confounded with the 
yellow tint sometimes assumed by the expectoration in 
summer time. This occurs in various diseases, and the tint 
is very similar to the yellow of an egg. The color is con- 
fined to the superficial layers of the mucus, and often 
becomes more pronounced after the expectoration has re- 
mained some time in the spittoon. It is dependent upon 
the presence of bacteria, which in their growth produce 
the pigment. It is a fact worthy of remark that not in- 
frequently several patients in the same ward exhibit the 
same coloration of the sputa, especially those in neighbor- 
ing beds. From similar causes a greenish tinge is some- 
times observed. 

Black-colored sputa may be said to be normal, 
for they are almost always met with in towns or 
manufacturing districts. The black hue is owing to the 
admixture of particles of carbon, dust, etc., which every- 
body inhales more or less. The particles are found partly 



254 MEDICAL MICROSCOPY. 

free and partly imbedded in cellular elements, hereafter 
to be described. 

In cases of malignant disease of the lungs the expectora- 
tion is generally tinged a dark red, and has a peculiar 
gelatinous consistency ; sometimes, however, both with 
carcinoma and sarcoma, various shades of green appear. 

Before quitting the consideration of the diagnostic 
value of the color of the sputa, a few words must be said 
about what is known as ^^ spurious haemoptysis; " that is 
to say, blood ejected from the mouth, but having its origin 
not from the lungs, but from the upper part of the air 
passages, the trachea, pharnyx, mouth, and nose. 

The diagnosis from lung disease, however, is more 
easily made clinically than by an examination of the con- 
tents of the spittoon. 

Hysterical patients often prick and suck their gums in 
order to elicit sympathy, and obtain credit for being the 
subjects of serious pulmonary disease. 

Dr. Douglas Powell (^' Diseases of the Lungs and Pleu- 
rae," 3d edit., p. 363, et seq.') divides false or spurious 
haemoptysis into seven divisions: — i. In cases of epistaxis 
the blood commonly trickles down the back of the throat 
and excites cough, by which it is removed in clots, stain- 
ing the saliva. 2. Ulceration of the throat, especially 
when malignant, may lead to copious hemorrhage. 3. 
Hysterical haemoptysis (already referred to). 4. A mor- 
bid state of the gums frequently arises from want of due 
attention to the teeth, or from the presence of decayed 
stumps in the alveoli ; blood may exude on the slightest 
friction. 5. An insufficient supply of vegetable food leads 
to a spongy, congested state of the mucous membrane of 
the mouth and fauces, very similar to the lesions character- 
istic of scurvy, and this is one of the most common causes 
of spurious haemoptysis. 6. In certain cases of anaemia 



EXAMINATION OF SPUTUM. 255 

the mucous membrane of the mouth and fauces exudes a 
sanguineous fluid. The transudation is very slow and in 
the daytime scarcely noticed, but during the night some 
accumulation takes place, and on waking the patient expels 
some bright red, unaerated fluid containing a few coagu- 
lated films, giving an appearance closely resembling that of 
currant jelly and water. 7. General hemorrhage from the 
whole mucous membrane of the mouth is sometimes seen 
in haemophilia. 

Odor. 

Inferences to be derived from the odor of the sputa are 
not of any material value. The chief exception is 
that of gangrene of the lungs, in which complaint the ex- 
pectoration has a peculiarly offensive and penetrating 
smell. 

In phthisis and other diseases in which the secretion is 
retained for some time in the air passages, it acquires a 
faint sweetish odor, due to decomposition. 

A few other naked-eye characters of the sputum remain 
to be considered. 

In the later stages of phthisis, when cavities have formed, 
the sputum becomes nummulated. It then consists of 
opaque, greenish discs, about the size of a sixpenny piece. 
If ejected into water these discs sink and spread out into 
irregular and ragged masses. They are probably formed 
by the passage of a thick secretion through the very fine 
bronchial tubes (Frankel). A similar condition is seen 
when an empyema has burst into the lungs (Traube). 

When some forms of sputa are allowed to stand they 
separate into three layers, the uppermost of which con- 
sists of semi-opaque, mucoid, and generally frothy liquid. 
The middle is a serous-like fluid, somewhat cloudy and 
whey-like ; whilst the undermost is made up of a mass of 



2s6 



MEDICAL MICROSCOPY. 



pus cells and debris, and is usually of a dirty yellowish 
color. This formation occurs most commonly in bron- 
chiectasis, and very typically also in putrid bronchitis and 
gangrene of the lungs. 

Fig. 48. 




Bronchial Stolon i^Bizzozero and Firket). 



In one of the rarest diseases known to physicians, 
^'plastic bronchitis," peculiar formations termed ^^bron- 
chial casts '' or *' stolons " (Fig. 48) are expectorated. 

The sputum in this disease is either of a catarrhal type, 



EXAMINATION OF SPUTUM. 257 

or contains a large proportion of blood. In it are masses 
covered with mucus rolled up in the form of balls and 
stained with blood. If these are thrown into water they 
spread out, and when freed from mucus are seen to consist 
of complete casts of parts of the bronchial tree. According 
to Biermer, they may extend to its finest subdivisions, so that 
the minute terminal filaments may show bulbous ends, due 
to their having been moulded in the infundibula. Their 
color is a grayish-white, sometimes, however, having a red- 
dish tinge. This depends either upon their being merely 
stained with blood externally, or owing to their central 
cavity being filled with blood clot. In the great majority 
of cases this central bore contains bubbles of air, produc- 
ing an appearance as of a string of pearls within them 
(Frankel). 

When examined more minutely the casts are found to 
be composed of a number of concentric laminae, separ- 
ated at intervals by narrow spaces, and with a central 
cavity running their whole length, except in the finest 
branches. When submitted to microscopic examination 
they are found to consist of a fibrillar or hyaline base, 
in which are imbedded large numbers of leucocytes. 
Charcot-Leyden crystals are often found in quantities 
within the meshes. The length of the casts is usually 
13^ to 2 inches, but sometimes they are much longer, 
even attaining 7 inches. Their diameter is narrow, 
rarely exceeding that of a goose quill. They divide 
dichotomously, the branches gradually diminishing in 
length and thickness. A slight bulging is often seen at 
the points of division, dependent on a similar condition 
in the branches of the bronchi themselves. According 
to Biermer, the site of formation of the casts may be 
inferred from the length of the intervals between succes- 
sive points of bifurcation, the short, rapidly branching 



258 MEDICAL MICROSCOPY. 

stolons being derived from the tubes of the upper lobe of 
the lungs, and the conaparatively longer tubes from the 
lower lobe. The masses expectorated at different times 
by the same patient are often so similar in size and ar- 
rangement as to give forcible corroboration to the sug- 
gestion that they have been produced in the same tubes. 

Similar bodies are found occasionally in pneumonic 
sputum, and in the rare cases in which diphtheritic 
membrane has penetrated to the finest branches of the 
bronchi. The casts in such a case are not so complete as 
in plastic bronchitis, and are thicker and less elastic. 

Casts very similar to the above are occasionally seen 
after an hemoptysis ; they are easily distinguished from 
the stolons, being homogeneous in structure, more trans- 
parent, and only consisting of a few branches, these 
being difficult to trace and to separate from one 
another. 

We need not in this little work enter into fuller details 
of the characters of sputa diagnostic of the different 
diseases of the respiratory organs, but will now pass to 
the 

Microscopic Examination. 

The same precautions must be taken in collecting the 
samples as were described in preparing for the macro- 
scopic examination, and especially is the sputum to be 
selected which is coughed up eaijy in the morning, 
before any food has been taken. 

As a rule special portions have to be picked out and 
placed beneath the microscope, and this process is greatly 
aided by emptying the spittoon into a black vulcanite 
dish, similar to those employed by photographers, or into 
dishes specially prepared for this work. By this means the 
opaque-white particles which yield the most useful infor- 



EXAMINATION OF SPUTUM. 259 

mation are rendered conspicuous and more easily recog- 
nizable. 

These portions are most conveniently removed by 
forceps and scissors ; it is a good plan to slightly bend the 
tip of one of the blades of the forceps, so as to form a 
small hook. Another method very generally adopted is 
to employ two steel pens, these act admirably, both as 
tearers and lifters. 

Some aid is rendered to the general microscopic exami- 
nation of sputum by pouring it into a large conical glass 
containing water, the heavier portions, such as fragments 
of lung tissue, spirals, pieces of membrane, etc., will fall 
to the bottom after a few hours, and may be removed by 
means of a pipette. 

In the preliminary examination any small opaque por- 
tions are removed and placed in the centre of a glass slide, 
and a cover-glass laid on the specimen, and gentle pressure 
applied in order to obtain a uniform layer. If it be desired 
to preserve the specimen a drop of glycerine must be added, 
then any superfluous moisture removed from the edge of 
the cover-glass by means of filter paper and liquid balsam 
run round its edges. Certain objects, such as leucocytes, 
pus cells, salivary corpuscles, etc., will be found in all sputa, 
and in others objects of more diagnostic value. 

We now proceed to describe in detail the various formed 
elements which may be met with. 

I. Leucocytes and Mucus Corpuscles (Fig. 49,/). 
— In greater or less numbers these are constanc ingredients 
of all sputa. The latter are less granular in appearance 
than the former, and are possessed of more nuclei ; they 
are larger too than the leucocytes, but owing to the effects 
of diffusion it is often almost impossible to distinguish 
between them. In mucoid expectoration the mucus cor- 
puscles naturally predominate, whilst in purulent sputa the 



2 6o MEDICAL MICROSCOPY. 

pus cells (leucocytes) may occupy the entire field. Both 
varieties of cells are seen in all stages of fatty degenera- 
tion, and may contain pigment granules, particles of 
carbon etc., and after an haemoptysis, crystals of haematoi- 
din. It will be obvious from these remarks that very few 
deductions can be with safety made from their presence. 
Closely resembling these cells, but rather larger, are the 
' ' salivary corpuscles, ' ' 

2. Compound Granule Cells (Fig. 49, <f).— These 
bodies are found in most sputa, often in so great numbers 
as to conceal all the other elements. In general appear- 
ance they resemble leucocytes, but are three or four times 
as large. In shape they are round or oval. Their proto- 
plasm is coarsely granular, and they contain several large 
oval nuclei which are rendered more conspicuous by the 
addition of a drop or two of dilute acetic acid. They are 
frequently the seat of particles of carbon and dust, and 
often appear in an extreme stage of fatty degeneration. 

Again, sometimes peculiar formations are found in them, 
first described by Virchow (*^ Archiv," Bd. vi, page 562), 
and termed by him ''myelin drops" (Fig. 49, d), 
owing to their similarity in appearance to coagulated nerve 
pulp. These objects are irregular in form, being some- 
times circular, sometimes spindle- and sometimes club- 
shaped, and having a tendency to flow together and form 
unshapely masses. They are also found floating free in the 
general mass of mucus. The chemical nature of these 
bodies is uncertain, but most authors consider that they 
consist of lecithin. 

The origin of the compound granular cells has been 
much disputed. The majority of observers ascribe their 
source to the alveoli of the lungs. Against this view is 
the fact that they are not only found in conditions in 
which the alveoli are affected, but also in bronchitis and 



EXAMINATION OF SPUTUM. 



261 



in the morning expectoration of perfectly healthy people. 
In favor of this view on the other hand, as before stated, 
is the fact that they are the principal seat of foreign par> 
tides which have been inhaled into the lungs, and are 
especially numerous in miners, etc., suggesting the idea 
that a slight irritation is set up, sufficient to loosen these 
cells, and so cause their presence in the pulmonary secre- 
tion. Other authors, of which Cohnheim is the chief, 
consider that they are simply lymph corpuscles which have 
become enlarged and swollen owing to the imbibition of cel- 
lular debris, etc. I most certainly incline to this last view. 




Various Cells in Sputum. 



Squamous, b. Columnar, c. 
ule cells, f. Leucocytes, g. 



Cubical, d. Myeloid, 
Red blood cells. 



e. Compound gran- 



3. Red Blood Cells (Fig. 49, g), — The subject of hae- 
moptysis has already been fully considered. The red 
corpuscles, contrary to what occurs in the urine, retain, as 
a rule, their shape and color. When they are very numer- 
ous they form rouleaux, and preserve their ordinary 
histological behavior. Under some circumstances, as in 
pneumonia, the corpuscles yield up their color to the 
general mass of sputum, and then appear as colorless 
rings, and the same process of destruction is also noticed 



262 MEDICAL MICROSCOPY. 

in gangrene of the lungs. In pneumonia they may only 
be represented by crystals of haematoidin. 

4. Epithelium. — Three varieties of epithelium are 
found in the sputa, namely, squamous cells derived from 
the buccal cavity, pharynx, and upper part of the larynx ; 
columnar cells from the greater portion of the respiratory 
passages ; and cubical cells from the terminal bronchi and 
alveolar walls. 

Squamous cells (Fig. 49, a) are always found, and there- 
fore have no pathological interest. When heaped together 
in big masses their edges are very likely to be mistaken 
for elastic fibres, but careful focusing will obviate this 
error. 

The columnar cells (Fig. 49, S) are of more value in 
diagnosis, for when they occur in large numbers they 
denote an inflammatory state of the parts from which they 
are derived. They are usually more or less altered, from 
their original form becoming swollen and pear-shaped, and 
their protoplasm more hyaline in appearance, and through 
partial loss of their contents form the well known ^^ goblet 
cells. ^' Although in their original state they are furnished 
with cilia, these appendages are very seldom preserved in 
the sputum. The cilia occur under only two conditions, 
namely, strong mechanical irritation of the bronchial 
mucous membrane, or ulcerative destruction of the same. 
From the first cause it is that they are sometimes seen in 
asthmatic sputum, for here the smaller bronchi are irritated 
by efforts to expel the thick and tenacious masses of 
secretion (Frankel). 

Cubical epithelium (Fig. 49, <f) is derived from the 
alveoli ; the cells are not often met with in their original 
condition, but are more or less altered by fatty degenera- 
tion. In the expectoration of patients suffering from 
brown induration of the lungs, especially in that ejected in 



EXAMINATION OF SPUTUM. 263 

the early morning, small reddish-brown streaks and parti- 
cles are found, which when examined microscopically are 
found to consist of masses of pigmented epithelial cells, 
the particles of pigment being circular or irregular, rarely 
crystalline. According to some authors the compound 
granule cells which have been just described are considered 
to be altered alveolar epithelium. 

5. Elastic Tissue (Fig. 50). — In the microscopic 
examination of sputum the search for elastic tissue stands 
second only in importance to that for tubercle bacilli. 
This tissue may have its source in the alveoli of the lungs, 
in the mucous membrane of the respiratory tract, or in 
the vocal cords and epiglottis. In the large majority of 
cases, however, it is derived from the alveoli. 

When present it is essentially diagnostic of partial de- 
struction of the lungs or other parts of the respiratory 
tract. A little experience will very soon enable the ob- 
server to recognize the fibres derived from the paren- 
chyma of the lung from those of the bronchi or larynx. 

In order to search for elastic tissue, the sputum for 
twenty-four hours should be collected and thrown into a 
black dish. Sometimes the collections of elastic fibres are 
so large that they appear as opaque, white specks floating 
in the mucus. When one of these is removed and placed 
under the microscope, it will be seen to consist entirely of 
large meshes of elastic tissue, showing quite plainly an 
alveolar arrangement, roughly tracing the forms of the 
alveoli. Such cases, however, are not common, and, as a 
rule, one must search in the general mass of expectora- 
tion for any particularly opaque particles, remove them 
by means of forceps and scissors, and place them on 
a slide. A cover-slip is next laid on, and gentle pres- 
sure used to obtain a uniform layer. The specimen must 
then be examined with a ^ inch lens, and after a little 



264 MEDICAL MICROSCOPY. 

practice the curled fibres will soon be recognized. By 
this method they retain exactly their original form, and 
much useful information may be gained from a study 
of them. If the sputum be very thick and tenacious, 
some aid in rendering it more transparent may be gained 
by running under the cover-glass a drop or two of a thirty 
per cent, solution of caustic potash. This dissolves the 
mucus and other matters, leaving the elastic fibres un- 
touched. Several preparations should be examined before 
a negative result is announced. 

Another method which is frequently practiced is one 
which was first introduced by Dr. Samuel Fenwick. It 
has the advantage of being more certain of detecting 
elastic tissue if present, but the appearance of the fibres 
is considerably changed, and many characteristics pre- 
sently to be alluded to are lost. After considerable 
experience I have come to the conclusion that the 
former method, although, perhaps, more tedious, is to 
be preferred. 

Dr. Fenwick' s method is carried out as follows: — The 
sputum is collected for twenty- four hours and poured into 
a beaker. An equal quantity of a solution of caustic 
soda, twenty grains to the ounce, is then added, and the 
mixture boiled until it becomes perfectly liquid, being 
occasionally stirred with a glass rod. When this stage 
has been reached the heating must immediately cease, 
for over- boiling will either dissolve the elastic tissue or 
render it so transparent and irregular in appearance that 
it can scarcely be recognized. When the boiled fluid 
has somewhat cooled it is thrown into a conical glass 
containing about four times its volume of water. In an 
hour or so a deposit will have formed in which the 
elastic fibres will be found, and portions of it may be re- 
moved with a pipette and examined under a microscope. 



EXAMINATION OF SPUTUM. 



265 



Elastic tissue is composed of fasciculated fibres, highly 
refractive, but possessing dark contours. They branch 
dichotomously. They have a slightly yellowish tinge, and 
have a tendency to break across, leaving a sharply defined 
edge. They usually occur in wide hoops and curves. 
Their clearly cut outline is especially characteristic, and 
enables them to be distinguished from other bodies, such as 

Fig. 50. 




Elastic Tissue. 



cotton and silk fibres, for which they might otherwise easily 
be mistaken. 

When derived from the parenchyma of the lung they 
will be seen to have a distinctly alveolar arrangement, as is 
well shown in the accompanying figure (Fig. 50), which 
was procured from the expectoration of a patient about 
twelve hours after an injection of tuberculin had been ad- 
ministered. 

When coming from the larynx the arrangement of the 
fibres is more linear, the fibres being straighter, and devoid 
23 



2 66 MEDICAL MICROSCOPY. 

of the bold curves which are so characteristic of the last 
named variety. 

Fibres from the other parts of the mucous membrane of 
the respiratory tract are similar to those coming from the 
larynx in their general arrangement, but are finer, and the 
bundles are generally of smaller dimensions. 

Their diagnostic value is great. Roughly speaking, 
their presence indicates that a destructive process is occur- 
ring somewhere in the respiratory tract. If from the larynx 
or bronchi there is probably considerable ulceration. If 
derived from the former the laryngoscope will give a far 
more satisfactory diagnosis than the contents of the 
spittoon ; but it is in those cases in which the arrangement 
of the fibres denotes that the parenchyma of the lung is 
affected that their recognition proves of so much value. 

It was at one time thought that they were diagnostic of 
tubercular disease of the lung. Troup (^^ Sputum; its 
Diagnostic and Prognostic Signification ") maintains that 
where the elastic tissue of the pulmonary alveoli is seen, 
the correct diagnosis in nine cases out of ten will be that 
of tubercular phthisis. If tubercle bacilli be also found, 
of course the diagnosis is assured, but the same author 
considers that careful microscopy of the expectorated 
matters can frequently detect its presence in a very early 
stage of phthisis, even when percussion and auscultation 
practiced by skilled observers give only doubtful or nega- 
tive results. Dr. Troup even goes further than this, and 
states that he has frequently found elastic tissue months 
before bacilli made their appearance. I cannot say this 
has been my experience, and would most certainly give 
the palm to the tubercle bacilli for early diagnostic worth. 

In addition to being found in phthisis, elastic tissue is 
occasionally seen in the sputum of pneumonia, even when 
the disease has run a normal course (v, Jaksch). The 



EXAMINATION OF SPUTUM. 267 

fibres are also found in cases of abscess of the lung, and 
they are then seen on careful examination to be thickly 
set with pus corpuscles, and to be grayish-yellow in color, 
especially at their edges. They are also found in the 
sputum from bronchiectatic cavities. 

In case of gangrene of the lung these fibres are again 
occasionally found. They are generally of a dirty color 
with jagged edges, and are surrounded by grayish-yellow 
masses. Their appearance in this disease is not very com- 
mon, in fact rather the exception, the reason probably 
being that though there is a considerable destruction of 
lung tissue the elastic tissue is dissolved by the ferments 
evolved during the process. Frankel gives a somewhat 
similar explanation, suggesting that the fibres are destroyed 
by the numerous bacteria which are present. 

In addition to the information yielded by the presence 
of elastic tissue of disintegration of lung tissue, more 
precise knowledge may be obtained by closer examination, 
as has been pointed out by Sir Andrew Clark. Thus if 
complete alveolar rings are present which are at the same 
time very elastic, we may assume that there is a rapid de- 
struction of lung tissue taking place. 

If small '' tailed " pieces only are seen, which have lost 
their elasticity, a more chronic process is indicated, whilst 
occasionally fibres are found encrusted with lime salts (Fig. 
51) and these must have lain for a considerable period in 
the lung cavity. 

Care must be taken not to mistake other bodies which 
are often present in the sputum, for elastic tissue; such 
objects are portions of fungi, as the Leptothrix buccalis 
and various forms of aspergillus. These are not so regular 
in outline as the elastic fibres and are generally surrounded 
by spores. A description of these fungi, will be given 
later on. 



268 MEDICAL MICROSCOPY. 

But the bodies which are most likely to mislead are 
fibres of cotton, silk or flax (see p. 278) ; a little practice, 
however, will soon prevent the observer from making any 
such blunders. Fibrillation of the mucin of viscid sputa 
will sometimes give rise to an appearance, at first sight 
closely resembling a bundle of fibres, but careful focusing 
will easily discern between the two. 

Fragments of Connective Tissue. — These only 
occur in rare cases and are derived from the walls of the 
alveoli and bronchi. They occur as dark gray particles, 
and are easily recognized. They are chiefly found in cases 

Fig. 51. 




Fragments of Elastic Tissue Partially Encrusted with Lime-Cells. 

of gangrene and abscess of the lung. In ulcerative pro- 
cesses affecting the larynx, small portions of cartilage may 
be loosened by the violence of the cough, and be ejected 
with the expectoration. 

7. Curschmann's Spirals (Fig. 52). — These peculiar 
bodies are chiefly found in the sputa of asthmatic patients, 
where they are sometimes exceedingly numerous. They 
were at one time considered characteristic of asthma, but 
have since been shown to occur, although only in small 
numbers, in the expectoration of pneumonia and of acute 
and chronic bronchitis. They were first minutely described 
by Curschmann, and hence their name. 



EXAMINATION OF SPUTUM. 269 

If the sputum from a case of bronchial asthma be poured 
out on a black plate, small opaque white particles will be 
seen which, even with a low-power lens, may be seen 
to consist of spiral formations. When one of them is 
placed under the microscope, an intricate arrangement 

Fig. 52. 




Curschmann's Spirals [T^'oup). 

of spiral threads can be discerned. In the centre there 
is a highly refracting thread, sometimes straight and 
sometimes twisted corkscrew fashion. Around this, in a 
complicated manner, is entwined an ensheathing layer 
of fine threads, in the meshes of which are seen leu- 
cocytes and epithelial cells. 



270 MEDICAL MICROSCOPY. 

Some of the spirals will be noticed to possess a peculiar 
prominence, and if these be more closely examined a large 
collection of pointed octahedral crystals of various sizes 
will be found. 

The exact structure of these spirals has not yet been 
definitely 'settled. According to some observers they con- 
sist largely of collections of bacteria, but the majority of 
authors consider that they are formed of mucus which has 
been moulded into spiral shape in its progress through the 
small bronchioles. The central core is due partly to an 
optical effect and partly to the greater compression which 
it has apparently undergone. 

Three modifications of the spirals are generally met 
with. Firstly, the complete spiral, consisting of the 
central thread surrounded by the mass of mucoid ma- 
terial ; secondly, the central thread alone is often seen, 
and, thirdly, the spirally twisted portion of mucus may 
occur without the central thread being visible. The 
diagnostic value of these bodies has not been established 
with any certainty. They are probably due to a catarrh 
of the finest bronchioles, and as before stated they are most 
commonly associated with bronchial asthma. 

8. Tonsillar Casts (Fig 53). — Casts of the crypts of 
the tonsil, or more frequently fragments of such casts are 
occasionally found in the sputum. 

They chiefly occur in connection with acute inflamma- 
tory diseases of the tonsils, but in comparison with the 
clinical aspects of the case have little diagnostic value. 
They bear considerable resemblance to masses of mucus, 
and are consequently easily overlooked, but they are more 
likely to be mistaken for small leashes of elastic tissue. 
Tonsillar casts appear as irregularly shaped bodies com- 
posed of parallel fibres finely knit together into a close net- 
work. When teased out the appearance represented in 



EXAMINATION OF SPUTUM. 



271 



the figure is produced. A complete cast assuming the 
exact form of a tonsillar crypt is rarely met with, but as 
already stated, more commonly only fragments are found. 
9. Corpora amylacea. — These bodies are very rarely 
met with ; they were first described by Friedreich in 
1856. They appear as oval or circular grains and are very 
similar in form to starch grains. They are composed of a 



Fig. 53. 




Cast of a Tonsillar Crypt (from a drawing kindly lent by Dr. Sheridan 

Delapine). 

vitreous looking substance, stratified in irregular layers 
around a central nucleus, which differs in appearance from 
the peripheral layers which are arranged so as to present a 
radiated formation. The central portion is sometimes 
scooped out so as to form a small hollow from which shal- 
low fissures extend, and occasionally is filled up with a 
material of different composition to the rest of the grain. 



2 72 MEDICAL MICROSCOPY. 

These amyloid bodies are characterized by their chemi- 
cal reactions. If a solution of iodine in iodide of potas- 
sium be run in under the cover-glass a deep blue color is 
produced, which becomes almost black on the addition of 
sulphuric acid ; the nucleus, however, is stained yellow by 
the iodine. Methyl violet causes the same reaction as in 
amyloid disease generally (see p. 107), coloring the grains 
red, whilst the surrounding tissues assume a blue tinge. 
Picro-carmine has a peculiarly selective action, staining 
the bodies a deep red. With methylene blue the corpora 
amylacea assume an emerald-green color. 

As regards the possible significance of these amyloid 
concretions many diverse theories have been suggested, 
the two principal being those of Friedreich and Langhans. 
The former considers that they are derived from remains 
of hemorrhoids in the lungs. In all his cases there was 
a history either of recent hemorrhagic infiltration, or 
traces of past inflammation. His theory is that a minute 
clot forms locally, thus constituting the nucleus, and fibrin 
is deposited in layers around it, and that this formation 
undergoes modification (of what kind is at present un- 
known) which causes the bodies to assume their character- 
istic appearance and properties, 

Langerhans on the other hand assigns to the corpora 
amylacea a cellular origin. In this view he has but few 
supporters, and, moreover, the bodies described by him 
do not agree in all particulars with those now under con- 
sideration. The observations of Jurgens and Zahn, and 
those more recently of Curtis, corroborate those of 
Friedreich. 

10. Portions of Membrane. 

a. Diphtheritic Membrane. — In cases of mem- 
branous laryngitis, whether occurring after scarlet fever 
or being undoubted instances of diphtheria, portions of 



EXAMINATION OF SPUTUM. 273 

the membrane are often coughed up and should be sub- 
mitted to careful microscopic examination. 

The membrane consists of a soft, thin, whitish layer, 
presenting fine striation and wavy lines, in the meshes of 
which are pus cells and epithelial debris. The fibrillated 
substance and cells alternate so as to produce a laminated 
structure. 

To demonstrate its characters it should be placed in 
water, when it can easily be spread out. 

Portions of it should be stained in methylene blue (see 
page 96), in order to ascertain whether any organisms be 
present. In the membranous laryngitis occurring after 
scarlet fever this is never the case. 

In diphtheria (it being assumed that '^ croup " and diph- 
theria are the same disease) the pathogenic organisms are 
often found, though not invariably so. They consist of 
bacilli of various shapes, the organism being polymorphous. 

b. Echinococcus Membrane. — Hydatid disease of 
the lung is an extremely rare one ; there are, however, 
several cases on record. The membranes are the same as 
those found in disease of the liver, and further description 
will be postponed until a future chapter. 

Occasionally the cysts themselves or loose booklets are 
expectorated, rendering the diagnosis easy. 

The source of these bodies, however, is not necessarily 
the lungs, as they may arise from a suppurating hydatid 
cyst of the liver perforating through the diaphragm into 
the lung. 

II. Crystals. — Many forms of crystals are found in 
the expectoration, but few of them have any diagnostic 
importance. 

a, Haematoidin. — These crystals occur in the form of 
ruby-red rhombic prisms or needles, often grouped together 
into bundles. Complete crystals are not often met with in 
24 



274 



MEDICAL MICROSCOPY. 



the sputum, haematoidin more commonly being found as 
irregular red particles. Both crystals and fragments are 
frequently enclosed in white blood corpuscles. 

Their presence indicates that blood has lain for some 
time in the air passages, or that an abscess has perforated 
into the lungs. They often occur in large quantities after 
an haemoptysis, or some time after a pulmonary infarct. 

If haematoidin is found chiefly enclosed in cells^ an old 
hemorrhage of the lungs is the most probable cause, but 

Fig. 54. 




.-mwi 



Charcot-Leyden Crystals. 



if the crystals are chiefly free there has most likely been a 
rupture of an abscess from some neighboring organ into 
the lung. 

^. Charcot-Leyden Crystals (Fig. 54). — In the 
sputum of asthmatic patients these crystals are often found 
in large numbers. They are long and pointed octahedra, 
or sharp or truncated fusiform bodies ; they vary greatly 
in size. They are soluble in alkalies, mineral acids, and 
warm water, but are insoluble in ether, alcohol and cold 



EXAMINATION OF SPUTUM. 275 

water. Their exact chemical composition is not known. 
Schreiner supposes them to consist of phosphoric acid 
combined with an organic base. Curschmann and Ungar 
are of the opinion that these crystals are derived from the 
decomposition of organic matter in the bronchioles. In 
connection with this, v. Jaksch's remarks are worthy of 
note ; he says that in fresh cases of bronchial asthma, or 
at the commencement of a new series of attacks, Cursch- 
mann's spirals are chiefly found, but no crystals; if such 
preparations are prevented from evaporating for about 
forty-eight hours, Charcot-Leyden crystals will be found to 
have been formed. In the further course of the attack the 
sputum when first ejected will contain the crystals. This 
seems to suggest that the crystals are formed by the 
decomposition of the spirals. 

They are not absolutely diagnostic of asthma, being 
present in other conditions. 

c, Cholesterin Crystals (Fig. 43, p. 214). — The well 
known rhombic, iridescent plates, with notches at one 
corner, are not often met with in sputum. 

The expectoration of phthisis, or the pus from a pul- 
monary abscess or empyema, are the most frequent condi- 
tions under which they occur. They generally lie together 
in heaps, are easily soluble in ether, but insoluble in acids, 
alkalies and water. When treated with dilute sulphuric 
acid and tincture of iodine, the crystals gradually change 
their color, becoming violet-blue, green and red. With 
sulphuric acid alone they slowly turn from yellow to violet- 
red. 

d. Crystals of the Fatty Acids. — These occur as 
groups of long, sharply-pointed needles, often arranged in 
small rosettes. They are characterized by their easy solu- 
bility in ether. 

They are most usually found in connection with putrid 



276 MEDICAL MICROSCOPY. 

bronchitis and gangrene of the kings, but also occur in 
the sputa of bronchiectasis and phthisis. They have 
therefore but little diagnostic value. 

As regards their chemical nature, they consist chiefly of 
palmitic and stearic acids, in combination with sodium, 
potassium, calcium and magnesium. 

e, Leucin and Tyrosin (Figs. 40 and 39). — These 
two substances are but rare constituents of the sputum. 
When present they in no way differ in form from that when 
appearing in urinary sediments. 

They have been described by Leyden in a case of putrid 
bronchitis, and in another in which an empyema burst into 
the lung. 

/. Oxalate of Lime (Fig. 37, p. 203). — These occur 
occasionally in their characteristic envelop-shaped 
crystals, or as an amorphous sediment. They have no 
diagnostic significance. 

g. Triple Phosphates (Fig. 41). — In appearance 
these crystals are the same as those described on p. 210. 
Being soluble in acids, they are only found when the 
sputum is alkaline. They sometimes occur in the sputa 
when a sanious exudation has broken into the lungs, and 
when decomposition of albuminous matters has taken 
place with evolution of free ammonia. 

12. Concretions. — It not uncommonly happens that 
small portions of cretaceous matter are coughed up by 
phthisical patients. When these particles are of a large size, 
they are known as ^' lung stones." Frankel also describes 
'^bronchial stones." These latter show indications of 
branching, and are of the size and shape which would be 
expected if they had been formed in the bronchi. 

The origin of both varieties is uncertain. Andral describes 
a case in which after death in a patient forty years old, who 
died of phthisis, he found a copious deposit of chalk in the 



EXAMINATION OF SPUTUM. 277 

cartilaginous portions of the smaller bronchi; the mucous 
lining was in some places ulcerated, and some of the cal- 
careous matter had escaped. Another source suggested is 
calcification of caseous matter which has been retained in 
a pulmonary cavity. Finally, some observers consider that 
these cretaceous particles are derived from bronchial glands 
which have become calcified and ulcerated through into the 
bronchi. As regards their size, this varies from that of a 
small pea to that of a plum -stone, or even of a walnut. The 
number ejected by one patient may be very great. Andral 
records a case of a phthisical patient who, in eight months, 
expectorated no less than 200 stones, the largest of which 
was the size of a cherry. 

13. Foreign Bodies. — For the most part these have 
little diagnostic worth. Some of them have evidently been 
inhaled, and again ejected, sometimes in so changed a con- 
dition as to be hardly recognizable. Such are particles of 
food which occasionally lie for a considerable period in the 
ventricles of Morgagni. 

Other instances of foreign bodies so expectorated are nut- 
shells, teeth, threads, etc. The retention of these bodies in 
the air passages often gives rise to serious symptoms, the 
explanation of which is only afforded on the expulsion of 
the offending body. 

Occasionally when malignant disease of the larynx is 
present, portions of tissue slough off and are ejected. Mi- 
croscopic examination of such fragments may yield most 
important aid in diagnosis and prognosis. 

Dr. Fagge records two cases in which hairs were expec- 
torated from mediastinal dermoid cysts. 

14. Accidental Ingredients. — In the opening part of 
this chapter stress was laid on the necessity of the preven- 
tion of extraneous matters from entering the spittoon. But 
sometimes in spite of all care such accidental substances 



278 



MEDICAL MICROSCOPY. 



will find their way into, and become mixed with the sputum. 
Most commonly these are particles of food matter, such as 
animal and vegetable tissues, globules of fat, starch grains. 



Fig 55. 




Silk Fibres. 



etc. Bundles of elastic tissue derived from the food may be 
distinguished from those coming from the patient by being 

Fig. 56. 




Cotton Fibres. 



coarser in texture, and arranged in a more parallel manner. 
Starch grains may be known from the corpora amylacea 
by their chemical reactions ; the former 'assume a purple 



EXAMINATION OF SPUTUM. 



279 



color when treated with iodine dissolved in iodide of 
potassium. 

Fig. 57. 




Wool Fibres. 



Fibres of silk, cotton, linen, etc., are also often met with. 
Fibres of silk (Fig. 55) are tapering, possess sharp out- 



28o MEDICAL MICROSCOPY. 

lines, and do not branch. Fibres of cotton (Fig. 56) are 
twisted, and have curious central markings. Fibres derived 
from linen (Fig. 57) are jointed, cylindrical, have ragged, 
uneven ends, and fine branching filaments at intervals. 
The fibres of wool (Fig, 58) are rounded, with fine cross- 
markings and reticulations in the border at the site of 
the cross-markings. The central longitudinal canal is 
usually invisible. Any of these fibres may be colored. 

To the above list must be added hairs, particles of dust, 
chips of wood from the floor, etc. 

Many of the above objects, unless carefully examined, 
may give rise to much misapprehension. 



CHAPTER XVII. 

THE MICRO-ORGANISMS OF SPUTUM. 

Probably no department of medicine has made greater 
advances during the last few years than bacteriology, and 
this has been largely made use of for clinical purposes in the 
examination of sputum. 

In this chapter will be described the most convenient and 
reliable methods employed for the detection of micro- 
organisms in the expectoration. 

I. Tubercle Bacilli. — A great number of processes 
have been from time to time introduced for the clinical 
detection of the bacillus tuberculosis in sputum. 

The method which long experience has proved to be 
speedy and reliable will be here described at length, short 
reference only being made to other processes. 

At the risk of some repetition of the directions given in the 
previous chapter, this method will be detailed throughout. 

In the first place the patient must be directed to reserve 
that portion of expectoration which has been ejected in the 
early morning before any food or drink has been taken. 
If nourishment has been partaken of during the night, it is 
well to request the patient to carefully rinse out his mouth 
with some water before using the clean spittoon. Nothing 
is more annoying than to have food matter mixed with the 
sputum. I have received samples so contaminated in this 
way that a satisfactory examination was out of ihe question. 
The collected sample is thrown out into a dish (preferably 
a dark colored one). Search is made for the small, yellow- 

281 



252 MEDICAL MICROSCOPY. 

ish, opaque, round particles so common in phthisical sputa, 
and if any are found they should be transferred to a cover- 
glass. Failing these, any other opaque portions must be 
selected. These having been placed on a cover-glass, 
another glass is laid on it, and the specimen of sputum 
gently spread out by means of pressure. A layer as thin as 
possible should be obtained, any excess that exudes from 
between the glasses being wiped away with a soft cloth. 

The cover-glasses are now separated by a sliding 
movement, that h, not ^'sprung" apart. Two films of 
sputum are thus obtained, which are allowed to dry in the 
air, this process being expedited, if desired, by holding the 
glasses between the finger and thumb over a spirit lamp, 
and as long as the heat does not burn the hand it will not 
injure the specimens. 

When dry the film must be fixed to the glass ; this is ac- 
complished by seizing the glasses one by one with forceps, 
and rapidly passing them three times through the flame of a 
spirit lamp or Bunsen burner, thus coagulating the albumin. 

A pause must now be made in the description of the pro- 
cess, to give the composition of the various fluids required. 
They are as follows : — 

Solution I. — Neelsen's solution (Ziehl). 

Fuchsine, I part, 

Dissolved in a 5 per cent, watery solution of 

carbolic acid, 100 parts. 

Alcohol, 10 ^' 

Solution 2. — Dilute watery solution of sulphuric acid, 
25 per cent. 

Solution 3. — Methylene blue. 

Methylene blue, 2 parts. 

Alcohol, 15 " 

Water, 85 " 



THE xMICRO-ORGANISMS OF SPUTUM. 283 

To resume, a small quantity of Neelsen's solution is 
placed in a watch glass, and heated until steam begins to 
rise. Into this the cover-glasses, prepared as previously 
described, are placed, film-side downward. It is some- 
times difficult to decide, especially in the later stages of 
the process, which is the prepared side of the cover-glass, 
but this can be determined either by lightly scratching 
each surface with a needle, and observing whether a mark 
is made, or more simply still by holding the cover-glass 
toward the light, when the side on which the film is ap- 
pears dull, whilst the unused side yields a bright reflex. 
The specimens are allowed to remain in the heated stain 
for two minutes. Most books state that five minutes are 
necessary, but experience has proved that two minutes are 
amply sufficient. 

In order to prevent the glasses sticking together, which 
they will do when both are submerged, the first glass should 
be placed slantingly in the fluid, so that it sinks. The 
second glass, however, should be held between the finger 
and thumb, and dropped on to the fluid. If this be done 
from a short distance, the glass will float. 

After a stay of two minutes in the stain the glasses are 
removed one by one, by means of forceps, and thoroughly 
decolorized in the dilute sulphuric acid contained in a shal- 
low capsule. A few seconds usually suffice for this, although 
contrary to what is generally believed, the bacilli retain the 
stain for at least thirty minutes in the acid ; they are in 
fact difficult to decolorize, and it is important that the red 
stain should be removed from other bodies as far as possi- 
ble, so that any excess should be on the side of a longer 
stay in the acid rather than the reverse. The best mode 
of procedure is as follows. The glass is retained in the 
forceps, and gently moved about in the acid, and after a 
few seconds is transferred to a dish of water. The color 



284 MEDICAL MICROSCOPY. 

will probably be partially restored, and the specimen must 
then be returned for a few seconds to the acid, again rinsed 
in water, this process being repeated until no more color 
is visible. 

The specimen is next placed, prepared side downward, 
in the methylene blue solution contained in a watch glass. 
It is allowed to remain for about half a minute, and is 
again washed in water. It is then allowed to dry, either 
by exposing it to the heat of the flame of the spirit lamp, 
or by gently pressing it between two folds of filter paper. 
It is finally mounted in water if only required for diagnos- 
tic purposes, or in balsam if a permanent preparation is 
desired. 

Thus prepared the bacilli (Frontispiece, Fig. i) appear 
as short red rods, whilst the mass of the sputum is stained 
blue. 

The tubercle bacilli are of variable size, but have an 
average length of half the diameter of a red blood cor- 
puscle. They are usually straight, but may be found 
slightly curved. Their grouping is very characteristic, 
being generally arranged in small numbers of two or three, 
lying cross-wise or at angles to one another. Sometimes, 
however, they are so numerous as to form large clumps, in 
which the individual members can only be recognized with 
difficulty. 

Their characteristic property is that of retaining the 
stain in spite of exposure to acid. By this they are dis- 
tinguished from all other bacilli, except that of leprosy, 
which in this country is hardly likely to be met with, and 
certainly never in expectoration. 

Their presence in sputum, therefore, is absolutely diagnos- 
tic of a tubercular process seated somewhere in the respira- 
tory tract, the exact locality being soon recognizable by 
the stethoscope or laryngoscope. 



THE MICRO-ORGANISMS OF SPUTUM. 285 

A peculiar beaded appearance is often noted, supposed 
by some to be due to the presence of spores which will not 
retain the stain. As regards their number, opinions differ 
considerably whether any definite conclusion as to the 
extent and activity of the disease can be drawn from ob- 
serving whether the bacilli are numerous or not. Dr. Theo- 
dore Williams considers that a large number of rods 
denotes rapid spread of the disease, but I am decidedly 
inclined to agree with Dr. Kidd and Mr. Taylor ('^ Trans- 
actions of the Royal Medico-Chirurgical Society," Vol. 
71, p. 173), who, after an exhaustive examination of the 
results at the Brompton Consumption Hospital, came to 
the conclusion that no importance, as regards prognosis, 
could be attached to the large or small number of rods 
seen in the specimens. 

A negative result is of little value and proves nothing ; 
but if, after repeated examinations of several satisfactory 
samples of sputum, no bacilli be found, this may be taken 
as presumptive evidence that the case is not a tubercular 
one, although it is no absolute proof. 

A positive result, on the other hand, is everything, and 
cannot be ignored. Although no other bacilli stain like 
the tubercle, yet errors sometimes arise, owing to foreign 
bodies retaining the color. Such matters as hair and horn 
are difficult to decolorize, and if the specimen has not re- 
mained sufficiently long in the acid, the edges of cells often 
appear red, but this mistake may easily be remedied by 
careful focusing. 

In order to examine satisfactorily for bacilli, an Abbe's 
condenser should be used, and, at the lowest, a one-sixth 
inch lens, preferably a one-twelfth, although they may be 
seen with a quarter. The method given above for staining 
tubercle bacilli can be thoroughly relied upon, but there 



286 MEDICAL MICROSCOPY. 

are several modifications of it and other methods, only a 
few of which will be considered here. 

A rather more rapid process may be conducted with the 
same stains. 

The cover-glasses are prepared in precisely the same 
way, but instead of being placed in a watch glass, a couple 
of drops of Neelsen's solution are placed on the prepared 
side of the cover-glass, which is seized in forceps. The 
glass is held over the flame of the spirit lamp until bubbles 
are observed to form ; the excess of stain is then poured 
off, and the glass swilled in the dilute sulphuric acid and 
washed in water. A drop or two of the methylene blue 
solution is then placed on the specimen, allowed to 
remain for half a minute, and washed off in water. The 
preparation is next dried and examined in balsam or 
water. 

According to Ehrlich it is very difficult to obtain the 
acid solution without free acid fumes, which tend to decol- 
orize the bacilli as well as the mucus. He therefore makes 
use of the following process: — 

The staining solution must always be prepared fresh. A 
few drops of pure aniline oil are placed in a medium-sized 
test-tube, which is filled with water, the mixture being 
thoroughly shaken and filtered through a double fold of 
paper. A little of the clear fluid is placed in a watch 
glass, and a few drops of a saturated alcoholic solution of 
fuchsine are added until the solution just becomes opaque. 
This being most easily judged by placing the watch glass 
on the edge of a piece of filter paper and observing when 
this edge becomes obscured. Cover-glasses are floated, 
prepared side downward, on this fluid for several hours 
(six to twelve). They are decolorized in a mixture of one 
part of nitric acid to two parts of a saturated solution of 
sulphanilic acid. 



THE MICRO-ORGANISMS OF SPUTUM. 287 

After being swilled in water and rendered quite 
colorless, by re-immersion in the acid solution, if necessary, 
they are counter-stained with methylene blue, washed in 
water, dried, and mounted in balsam. The rationale of 
this process is that the sulphanilic acid absorbs the 
nitrous fumes, so allowing the pure acid to act on the 
bacilli. 

The process recently introduced by Dr. Gabbet seems 
to be an excellent one. It closely resembles those already 
described, but is more rapid ; on the other hand one has 
not the same control over the process of decolorization. 

The first staining solution has the following compo- 
sition : — 

Carbolic acid (5 percent.), 100 c.c, 

Magenta, I gramme, 

Spirit, 10 c.c. 

Some of this solution is placed in a watch glass, the 
cover-glasses immersed in it and heated until steam 
begins to arise. They are then placed for one or two 
minutes in a solution, which at once decolorizes and 
counter-stains them. This solution is made as follows : — 

Sulphuric acid (25 per cent.), . 100 c.c, 

Methylene blue, ... 2 grammes. 

On being taken out the specimens are washed in water 
and mounted in the usual way. 

In some cases in which no bacilli are found by the 
above methods, but where the history and physical signs 
point to a tubercular process in the respiratory tract, 
Biedert's method (^^ Centralblatt flir Klinische Medicin,'* 
1 89 1, No. 6) should be adopted. 

To a tablespoonful of the sputum seven or eight drops 
of caustic soda are added, and two tablespoon fuls of 
water. This mixture is boiled until it becomes liquid. 
Four to six tablespoonfuls of water are now added, and 



288 MEDICAL MICROSCOPY. 

the mixture again boiled until the whole is a thin uni- 
form liquid. It is next thrown into a conical glass and 
allowed to stand from twenty-four to forty-eight hours. 
Part of the sediment which results is removed with a 
pipette to cover-glasses, spread out in as uniform a layer 
as possible, dried, and then stained for tubercle bacilli by 
one of the methods already detailed. 

The only other process for the detection of tubercle 
bacilli that will be described here is the one introduced 
by Dr. Heneage Gibbes. It is very simple and rapid, but 
in the opinion of the author not so reliable as the fore- 
going. Dr. Gibbes has a special staining fluid prepared 
as follows : — 

Rosaniline hydrochlorate, 2 parts, 

Methylene blue, i part. 

The mixture is triturated, and three parts of aniline oil 
dissolved in fifteen parts of rectified spirit is slowly added, 
and finally fifteen parts of distilled water. 

Cover-glasses are prepared in the usual way and im- 
mersed in the stain, which has been heated until steam 
has arisen, for five minutes. They are next washed in 
methylated spirit until no more color can be extracted, 
when they are dried and mounted. 

2. Pneumococci (Fig. 59). — In 1883 C. Friedlander 
announced that he had found the pathogenic organism 
of pneumonia. In the rusty sputum of pneumonia cer- 
tain organisms are almost always found, sometimes in 
very large numbers. Unfortunately, however, for their 
diagnostic value, micro-organisms, morphologically pre- 
cisely similar to them, are found in many other diseases, 
and indeed in the saliva of healthy individuals. 

These micrococci take the form of spindle-shaped bodies 
united at their flat bases, so forming diplococci. Some- 
times three or four are joined together. But what especi- 



THE MICRO-ORGANISMS OF SPUTUM. 



289 



ally characterizes them is the capsule with which they are 
surrounded ; they thus appear as oval bodies provided with 
a sheath of similar shape. 

There are two principal methods by which they are 
stained. 

Cover-glass preparations of the expectoration are pre- 
pared in the same way as was described when treating of 
tubercle bacilli. 

A few drops of aniline oil are placed in a test-tube, 
which is filled up with water and thoroughly shaken, the 

Fig. 59. 




Bacterium pneumonia crouposa, from Pleural Cavity of a Mouse X 

1500. 

A, B. Thread-forms. C, D, E. Short rod-forms. G. Diplococci. H. Cocci. 
/. Streptococci. {After Zopf, from Crookshank^ 

emulsion being filtered through a double fold of filter 
paper. A few drops of a saturated alcoholic solution of 
gentian-violet are added until the mixture becomes opaque. 
The cover-glasses are immersed in this solution for five 
minutes, and are then swilled for a few seconds in absolute 
alcohol, washed in water, and mounted in the usual way. 

Very satisfactory specimens may also be prepared as 
follows : — 

The aniline water is made as above, but instead of the 
25 



290 MEDICAL MICROSCOPY. 

violet a few drops of a concentrated alcoholic solution of 
fuchsine are added. The cover-glasses are immersed in 
this stain for about a minute, and are then placed for two 
minutes in a watery solution of methylene blue. Finally, 
they are washed in water, dried, and mounted in balsam. 
Stained in this way the diplococci are colored blue, the 
capsule assuming a rose tint, whilst the ground substance is 
a bluish-red. 

A. Frankel found a coccus precisely similar in appear- 
ance to the above, not only in the sputa of pneumonia, 
but also in other diseases, and even in normal saliva. I 
have found very characteristic specimens in the sputa of 
phthisical patients. It is conceivable that these organisms 
are normally present in saliva and only excite pneumonia 
when other causes combine to produce a favorable pabulum 
upon which they may grow, and evolve the poisons which 
are necessary to cause the conditions found in the disease. 
Franker s coccus and its capsule stain readily in methylene 
blue. After cover-glasses have been prepared in the 'usual 
way they are immersed in methylene blue solution for five 
minutes and then washed in water acidulated with acetic 
acid. 

3. Actinomyces. — This disease has been very rarely 
diagnosed in this country during life. Dr. Douglas Powell 
read a paper before the Royal Medico-Chirurgical Society 
(see ^^Transactions," vol. 72, p. 175) on a case which 
occurred at the Brompton Consumption Hospital. 

Very seldom, however, in any country has the charac- 
teristic fungus been found in the sputum. Baumgarten, 
Paltauf, and others have recorded such cases. Possibly if 
the sputum from obscure cases was more frequently ex- 
amined, more cases could be added to the list. The ex- 
pectoration in actinomycosis is usually small in amount, 
muco-purulent, often of putrid odor, occasionally stained 



THE MICRO-ORGANISMS OF SPUTUM. 29 1 

with blood, and contains minute white or yellowish gran- 
ules about the size of a small pin's head. When one of 
these granules is examined with a quarter-inch lens, a 
number of small oval bodies (clubs) are seen surrounding 
an indistinct mass, but to bring out clearly the appearance 
of the fungus, specimens must be stained in the following 
manner. A few of the granules are placed on a cover- 
glass, allowed to dry, and passed three times through the 
flame. It is stained by Gram's method (see p. loi), and 
afterwards counter-stained by immersion for twenty-four 
hours in a saturated watery solution of safranin, containing 
two per cent, of aniline oil, then cleared in xylol, dried, 
and mounted in balsam. The clubs of the fungus are thus 
stained yellowish-red, and the threads blue. Instead of 
the safranin, alcohol containing a little picric acid may be 
added after the Gram's process is complete, a stay of five 
minutes being necessary ; the clubs are then stained a 
bright yellow color. 

4. Micrococcus tetragonus. — This peculiar organ- 
ism, as its name suggests, consists of four cocci united 
sarcina-like, and surrounded by a hyaline capsule. The 
cocci are stained by all the aniline dyes, and are frequently 
seen in sputum when this has been stained by methylene 
blue. The capsule, however, does not stain. 

This microbe is not pathogenic, being found in the 
sputa of various diseases, or even in the expectoration of 
healthy people. It is most frequently seen in phthisical 
sputum. According to Koch, it assists in the destructive 
process taking place in the lungs. 

Gaifney showed that when injected into guinea pigs and 
white mice, the animals died of septicaemia. 

5. Septic Micro organisms. — Various forms of 
micro-organisms, both rods and cocci, are found in all 
sputa, being especially numerous in the expectoration of 



292 



MEDICAL MICROSCOPY. 



bronchiectasis and gangrene of the lungs. They vary in 
size, and take the forms of staphylococci, streptococi, etc. 

Diplococci are especially common, and are usually sur- 
rounded by a thin colorless zone. 

Occasionally zoogloea masses occur, made up of large 
numbers of micrococci. These are especially common in 
the sputum of phthisis. 

6. Fungi. — Several forms of fungous growth occur in 
the mouth, and are consequently not unusually found in 

Fig. 60. 







Oidium albicans (v. JakscJi). 

the sputum. More rarely they grow in the deeper respira- 
tory passages or even in the lungs themselves. 

The most common is the Oidium albicans, or thrush 
(Fig. 60) ; it occurs in the form of branching threads, 
composed of elongated cells, amongst which numerous 
spores are seen. They stain with methylene blue. The 
Leptothrix buccalis (Frontispiece, Fig. 2) often grows 
round the teeth, and portions being detached and appear- 
ing in the sputum may be a source of difficulty as regards 



THE MICRO-ORGANISMS OF SPUTUM. 293 

recognition, unless the observer be familiar with their ap- 
pearance. Its reaction with iodine is characteristic. If a 
little of the tincture of iodine, or of a solution of iodine 
in iodide of potassium be added to the specimen, the 
threads of the fungus assume a deep violet color, which, 
however, only lasts a few hours. 

The Aspergillus fumigatus has also been described 
by Virchow as occasionally growing in the lung. 

In some cases of extensive destructive ulceration in the 
lung, especially in pulmonary gangrene, bodies known as 
*' Sarcinse pulmonis,'* are found in the sputum. They 
were first described by Virchow and Friedreich ('^ Vir- 
chow's Archiv," ci, 401, 1856, and 390, 1864). In 
appearance they closely resemble the Sarcinae ventriculi, 
but are rather smaller. 

The only case in which I have found these bodies, was 
one of phthisis running a slow course, the physical signs 
being obscure, but tubercle bacilli were present in the 
sputum in large numbers. 

7. Infusoria. — These have been described by Kannen- 
berg (^* Virchow's Archiv," Ixxv, 471, 1879) ^^ ^^^ 
sputum of patients afflicted with pulmonary gangrene. 
They were chiefly found enclosed in small yellow droplets 
surrounded by fatty needles. They were of the monad 
and cerco-monad varieties. They exhibited sluggish move- 
ments. 

According to v. Jaksch the best method of demon- 
strating them is as follows. A few of the droplets are 
picked out and placed upon a cover-glass. About three or 
four drops of a one per cent, solution of common salt are 
added. The preparation is dried in the air and stained 
in a watery solution of methyl violet, washed in water, and 
while still wet placed in a concentrated solution of acetate 



294 MEDICAL MICROSCOPY. 

of potash. By these means the protoplasm of the monads 
is colored a beautiful blue. 

8. Entozoa. — Members of this group are very seldom 
found in the sputum. In the rare instances of hydatids in 
the lungs, the hooklets or even vesicles may be discovered. 

The Ascaris lumbricoides has occasionally found its way 
into the oesophagus from the intestines, and has been 
ejected, and Manson has recorded cases in which the eggs 
of Bilharzia haematobia have appeared, having escaped 
from the parent worm in the blood-vessels. 



CHAPTER XVIII. 

EXAMINATION OF VOMIT. 

An investigation by the microscope of matters which 
have been vomited does not yield such useful information 
as a similar examination of sputum, or even of the faeces. 
But occasionally considerable aid to diagnosis is rendered 
by such means, and the student ought to make himself 
familiar with the appearances which are met with when 
food has been partially digested. For this purpose he 
should soak pieces of bread, meat, tendon, cartilage, and 
the more common vegetables (such as potatoes, cabbages, 
etc.) in slightly acidulated water, and then tease out parti- 
cles of these substances on a glass slide and examine them 
with the microscope. 

Much more valuable information is gained from a chem- 
ical examination, especially as regards the detection of 
poisons, but the nature of this work forbids me entering 
into this subject here. 

The vomited matter is placed in a conical glass and 
allowed to stand for a few hours, when portions of the 
sediment should be removed with a pipette, placed on 
glass slides, protected with cover-glasses, and investigated 
with an inch and quarter-inch lenses. Separate portions 
of the supernatant liquid should also be examined. 
The following particles of food matter will probably be 
seen : — 

I. Muscle fibres (Fig. 6i, ^), recognized by the transverse 
striations. 

295 



296 



MEDICAL MICROSCOPY. 



2. Elastic fibres, characterized by their clear outlines 
and tendency to form curves. 

3. Fat globules and needles. The nature of these bodies 
may be proved by their solubility in ether. 

4. Vegetable cells of various forms. 

5. Starch granules (Fig. 61, ^), more or less altered by 
the process of digestion, but stained blue by the action of 
iodine dissolved in iodide of potassium, a few drops of 
which should be run under the cover-glass. 

Epithelium (Fig. 61, a). — Two varieties of epithelium 
may be noticed. Squamous cells occur in small numbers 




a. Epithelial cells, b. Muscle fibre, c. Starch granules, d. Sarcinse. e. Yeast 
cells, y. Non-pathogeni€ micro-organisms. 



only, and are derived from the mouth and other parts of 
the oesophagus, having been swallowed with the saliva. 

Columnar cells are sometimes very numerous and then 
are indicative of catarrh of the gastric mucous membrane. 
They are generally more or less altered in shape by the 
action of the secretions. In the vomit of cholera numer- 
ous small white flocculi are found, which may be shown to 
consist of collections of epithelial cells, both squamous and 
columnar. The occurrence of so-called ^^ cancer cells," 
in cases of carcinoma of the stomach, is doubtful. No 



EXAMINATION OF VOMIT. 297 

doubt cells derived from tumors do make their appearance 
in the vomit when such conditions exist, but they cannot 
with certainty be distinguished from squamous epithelial 
cells. If large, irregularly-shaped cells are found in large 
numbers, together with red blood corpuscles and pigment, 
this would be presumptive evidence that cancer of the 
stomach did exist, although without accompanying clinical 
signs, which are far more constant and valuable, such a 
diagnosis could not be ventured on. 

Red Blood Cells. — The appearance of red blood cells 
in the vomit depends upon how long they have been re- 
tained in the stomach. If a vessel has ruptured and a 
considerable quantity of blood has been poured out, they 
will present their normal color and biconcave form. As a 
rule, they are more or less altered by the action of the 
gastric juice, and are then seen as colorless rings. If the 
blood has been some time in the stomach the haemoglobin 
becomes reduced to haematin ; a dark brown fluid then re- 
sults, in which masses of pigment may be seen, recogniz- 
able as blood only by means of the spectroscope. 

Leucocytes. — A few scattered white blood cells are 
always present. If they occur in large numbers there is 
probably gastric catarrh, but the cells may be derived from 
the saliva, sputum, or nasal mucus which has been swal- 
lowed. These cells are seldom present in such quantity 
that the term pus may be applied, and practically this only 
happens when an abscess, situated in neighboring parts, 
has burst into the stomach or oesophagus. The cells 
themselves are generally so much altered by the secretions 
that only their nuclei are visible. 

In cases of diphtheria portions of membrane may be- 
come detached and ejected by the effort of vomiting. Their 
nature may be demonstrated by floating them in water, 
when they will easily spread out. 
26 



298 MEDICAL MICROSCOPY. 

Vegetable Parasites. — Various kinds of fungi are 
occasionally found in the vomit, some such as leptothrix 
have evidently been swallowed, and only two are of any 
importance. 

Sarcinse (Fig. 61, d), — These bodies appear as round 
cells grouped together in fours, and very much resemble 
wool-packs in form. They are of dark silver-gray color, 
and are stained mahogany-brown with a solution of iodine 
in iodide of potassium. They are generally associated 
with obstructive disease of the pylorus, so that food is 
retained and undergoes fermentation. 

Yeasts (Fig. 61, e). — v. Jaksch (/. c, p. 108) describes 
three forms of yeasts as occurring in the vomit. 

{a) Saccharomyces cerevisise. They are about the 
size of leucocytes and cohere in small groups. If a solu- 
tion of iodine in iodide of potassium be run under the 
cover-glass, they stain a brownish-yellow. These bodies 
are occasionally elliptical in shape. 

(^) Very small yeast-like fungi in thick clusters. 

(f) Rod-like bodies, highly refracting, and of consid- 
erable length and thickness. They have rounded extremi- 
ties, and occur separately or in strings. It has been stated 
that these are the agents in lactic acid fermentation of 
sugar. 

Micro-organisms (Fig. 61, /) are very frequently 
present in varying numbers. They take the form both 
of rods and cocci, but are non-pathogenic and therefore 
of no diagnostic worth. 

Animal Parasites. — These are purely accidental, and 
have entered the stomach from the intestines. The only 
ones which have been described are the Ascaris lumbri- 
coides, Anchylostoma duodenale, and Oxyuris vermicu- 
laris. 



CHAPTER XIX. 

EXAMINATION OF DISCHARGES AND CONTENTS OF 
CAVITIES. 

The vast proportion of specimens which medical men 
are called upon to examine with the microscope, are de- 
rived either from the sputum or urine. But occasionally 
they have to investigate deposits of fluids drawn from the 
chest or abdomen, or the discharges from abscesses, fistulse, 
etc. These materials, though hitherto commonly neg- 
lected, may yield valuable assistance in diagnosis, and 
the student therefore should make himself acquainted with 
at least the most important of such fluids, whenever occa- 
sion offers for their study. 

The fluids will either have been drawn off by puncture, 
or in some other way, or a discharge may have occurred 
spontaneously. If sufficient quantity of the material can 
be obtained, it should be placed in a conical glass, and 
allowed to settle for some hours, when the supernatant 
liquid can be poured off, and portions of the sediment re- 
moved by means of a pipette for investigation. Unstained 
specimens should always be examined, and subsequently 
such staining processes as experience may suggest should 
be applied. 

Serous Exudations. 

These resemble in general appearance blood serum. 
They are almost clear, have a pale yellow color, and coagu- 
late on standing, yielding a clot which is rich in fibrin. 
As a rule there is but very little sediment, and when 

299 



300 MEDICAL MICROSCOPY. 

placed under the microscope in the usual way, it is often 
very difficult to make sure that one is focusing the layer of 
fluid between the glasses. A few red corpuscles will usually 
be seen, and they generally preserve their form and color. 
Leucocytes are usually more numerous. Some endothelial 
cells derived from the surface of the serous membranes 
will be seen, but these are also scarce. In addition to 
these, Bizzozero (^^ Microscopie Clinique," p. 115) de- 
scribes some larger cells, varying in diameter from seven 
to thirty 11 ; they contain small gray droplets, which are 
sometimes so abundant that the cells appear to be entirely 
made up of them. These bodies seem to undergo a kind 
of cystic degeneration, so that large vacuoles are formed, 
containing a clear fluid. Micro-organisms are frequently 
present, chiefly micrococci, and large numbers may be 
present, although the fluid is quite clear. 

In one specimen which I examined, the liquid was yel- 
lowish in color and perfectly clear, with the exception of 
a few small white flakes. Yet tubercle bacilli were found 
in large numbers, especially in the flakes just mentioned. 
In order to search for the organisms, a little of the fluid is 
distributed over several cover-glasses, which are then ex- 
amined in precisely the same manner as films of sputum 
(see p. 282). 

If the serous fluid is of old standing, cholesterin crystals 
are generally seen. 

Hemorrhagic Exudations. 
Such liquids are of a brownish or red color, due to the 
presence of blood. In the deposit red blood corpuscles, 
more or less altered in color and appearance, are seen in 
large numbers, but occasionally the haemoglobin seems to 
have been dissolved out, and then only a few colorless 
rings are visible. Endothelial cells, often with advanced 



EXAMINATION OF DISCHARGES. 3OI 

fatty degeneration, are likewise to be found. In regard 
to these bodies, Quincke (^^ Deutsches Archiv flir Klin. 
Med.," 1882, 569) asserts that when treated with a solu- 
tion of iodine in iodide of potassium, a marked glycogen 
reaction is often produced, and that the probable presence 
of carcinoma is then indicated. Should they be accom- 
panied by large epithelial cells, the diagnosis of cancer 
may safely be made. 

Chylous Exudations. 
Beyond the appearance of particles of fatty matter no 
characteristic bodies can be found in the deposit of the 
fluid in such conditions. 

Sero-purulent Exudations. 
These often occur in tubercular cases, and the pathogenic 
organisms can be found without difficulty. Pus cells are of 
course numerous, but otherwise no special characteristics 
can be mentioned. 

Putrid Exudations. 
These are usually brownish in color, and are at once rec- 
ognized by their penetrating and disagreeable odor. The 
deposit is peculiar, exhibiting in addition to shrunken red 
and white blood cells, numerous crystals, chiefly fatty 
needles, cholesterin and haematoidin. These fluids swarm 
with septic micro-organisms. 

Purulent Exudations. 
If pus be allowed to stand in a cool place it separates into 
two layers, the upper of which is almost transparent, whilst 
the lower is whitish and opaque. A few streaks of blood 
are almost always present. When fresh, pus is of a grayish 
color, but when putrid it appears like a thin green fluid 



302 MEDICAL MICROSCOPY. 

with an extremely strong and unpleasant odor. If much 
blood be present the color depends upon the proportion of 
the two fluids. The discharge from hepatic abscesses has a 
peculiar chocolate color. 

In the sediment of a purulent exudation pus cells of 
course almost fill the field ; if perfectly fresh, amoeboid 
movements may be seen, and if a solution of iodine in 
iodide of potassium is run under the cover-glass, the corpus- 
cles assume a mahogany color. If the discharge is old, 
white cells, more or less shrunken and granular in appear- 
ance, are seen. Cells containing fat will also be noticed. 
Red blood corpuscles are usually present, sometimes in very 
large numbers. 

Crystals are seldom absent, the most common being 
needles of the fatty acids, cholesterin, haematoidin, and 
triple phosphates, but no special significance can be attached 
to their presence. 

The search for micro-organisms is of the greatest im- 
portance ; this is obvious, from the fact that the formation 
of pus has been shown to be almost entirely due to the action 
of bacteria, for if these be excluded from entering the 
fluids of the body, as is now accomplished by antiseptic 
surgery, a purulent process never occurs. Certain organ- 
isms therefore are peculiar to pus ; these are various forms 
of micrococci They are more numerous in old than in 
fresh pus, although they can also generally be detected in 
the latter. No fewer than eight varieties of micrococci have 
been separated from pus, but the most common are the 
following : — 

Staphylococcus pyogenes aureus.— These are cocci 
occurring singly, in pairs, in very short chains, and in 
irregular masses. Their presence causes no odor in the 
pus. They are found in the discharge from boils, and in 
the abscesses of pyaemia and puerperal fever. 



EXAMINATION OF DISCHARGES. 303 

Staphylococcus pyogenes albus. — Morphologi- 
cally, these are indistinguishable from the above and occur 
under similar conditions, but differ somewhat in cultiva- 
tions. Various pathogenic organisms are found in puru- 
lent discharges, and these are of the utmost importance, as 
their detection will be a most valuable guide to the surgeon 
or physician in the future treatment of the case. 

Tubercle Bacilli. — The method of detecting these 
organisms is the same as that described in the chapter on 
sputum. The layer of pus on the cover-glasses must be 
made as thin as possible, and to avoid errors it is import- 
ant to allow the glasses to remain in the acid until the red 
fuchsine is entirely got rid of. Ten minutes or a quarter 
of an hour are not at all too long. The lesions in which 
the detection of tubercle bacilli is of great importance are 
many, amongst which may be mentioned tubercular nodules 
of the skin ; I have seen two or three cases in which so- 
called '' post-mortem warts '* were proved to contain bacilli. 

Discharge from the ear in cases of tubercular meningitis 
in children is another example. 

Actinomyces. — This is a rare disease, although cases 
seem to have been more numerous during the past few 
years, probably owing to our improved means of investi- 
gation, and partly, perhaps, because a systematic exam- 
ination of purulent discharges in doubtful cases is more 
frequently made, than was formerly the case. It should 
be laid down as a rule that when a collection of fluid is 
diagnosed, if the nature of the disease is doubtful, and es- 
pecially if the discharge is purulent, a microscopic exam- 
ination should always be made. 

In actinomycosis the pus is thin and viscous, and will be 
seen to contain small nodules of a grayish color. If one 
of these be placed under the microscope, the characteristic 
club-shaped bodies will be seen. 



304 MEDICAL MICROSCOPY. 

Cover-glass preparations should be made in the usual 
way and stained by the method described on p. 106. The 
central mycelium will then be stained purple and will be 
seen to consist of closely interwoven threads surrounded by 
the radiating mass of clubs. This appearance is unique, 
and the *'ray fungus" cannot be mistaken for any other. 
In a case under Dr. Douglas Powell at the Brompton Con- 
sumption Hospital Mr. Taylor recognized the fungus in the 
discharge during life ; and Prof. Crookshank undertook an 
exhaustive inquiry into the nature of the parasite, the 
results being published in the Medico-Chirurgical Trans- 
actions, Vol. Ixxii, p. 193. He showed that the fungus 
appeared to be closely related to the Basidio-mycetes, and 
was able to demonstrate that the threads of the mycelium 
pass directly into the narrow central ends of the clubs. 

Bacillus of Leprosy. — The nodules which appear on 
various parts of the skin and mucous membrane in leprosy 
occasionally ulcerate, discharging a considerable quantity 
of thin pus. If cover-glass preparations of this discharge 
are made in the usual way, and stained in the same manner 
as sputum is stained for tubercle bacilli, numerous fine 
slender rods will be seen, hardly distinguishable morpho- 
logically from the tubercle bacilli. They stain, however, 
more readily, and unlike the bacilli of tubercle are not 
colored by Kiihne's method (see p. 98). 

Bacillus of Anthrax. — In this country two affections 
are produced by this organism. ^'Malignant pustule," 
due to a direct inoculation, and consisting of a local in- 
flammation, followed by the development of a brawny 
swelling; *^ wool-sorter's " disease, resulting from inhala- 
tion, or swallowing of the spores, so that the pulmonary or 
intestinal mucous membranes are first affected. 

In order to demonstrate the pathogenic micro-organism, 
the local affection or c.ubuncle should be incised and 



EXAMINATION OF DISCHARGES. 305 

cover-glass preparations made of the discharge. When 
stained by Gram's method, the bacilli can be well seen as 
straight rods, sometimes curved. Their extremities are 
sharply rectangular, and the bacilli are usually joined to- 
gether in chains, the segments being marked off by clear 
linear spaces. In sections the organisms should be stained 
by Weigert's modification of the Gram method, as described 
on p. 102. 

Bacillus of Syphilis. — The pathogenic character of 
this organism has not met with general acceptance. It was 
first described by Lustgarten. In form it somewdiat resem- 
bles the tubercle bacillus, but it is always found in the in- 
terior of nucleated cells about twice the size of leucocytes. 
They have been found in the discharge of the primary 
lesion and in hereditary affections of tertiary gummata. 
Cover-glass preparations of the pus should be stained in 
the following way : — 

A fresh solution of gentian aniline violet is made (see p, 
1 01) in which the sections are placed for from twelve to 
twenty-four hours. They are then removed and washed 
for a few minutes in absolute alcohol, and afterwards im- 
mersed for ten minutes in a 1.5 per cent, solution of per- 
manganate of potash. Next they are rinsed in a watery solu- 
tion of pure sulphurous acid, and finally washed in water. 
If by these means the preparations are not completely 
decolorized, they must again be washed for a few seconds 
in permanganate of potash, and then again in sulphurous 
acid and water, the process being repeated until no more 
color is visible. This method is not very satisfactory, as it 
stains other bacilli, as well as those of syphilis, and the 
whole matter requires further research before it can be 
determined that the characteristic organ of syphilis has 
been found. 

Another method has been introduced by De Giacomi, 



3o6 * MEDICAL MICROSCOPY. 

which consists in staining cover-glass preparations in ani- 
line fuchsine made in the same way as methyl aniline 
violet. The stain is heated and the glasses allowed to 
remain in it for about ten minutes, after which they are 
washed in water containing a few drops of perchloride of 
iron. They are next decolorized in a strong solution of 
perchloride of iron and counter-stained in Bismarck brown. 
The bacilli are then colored a deep red. 

Gonococcus. — The discharge obtained from patients 
suffering from gonorrhoea may be shown to contain a dis- 
tinctive micro-organism in the form of micrococci, occur- 
ring either singly, or more usually in groups of ten or 
twelve. Cover-glass preparations of the pus are stained in 
methylene blue and then thoroughly washed in water. 

Bacillus of Glanders. — The bacillus mallei is morpho- 
logically very similar to the tubercle bacillus. It may be 
found in the pus derived from the ulcerated nasal passages 
of patients affected with this disease. The organisms are 
difficult to stain. The process introduced by Loffler is 
the best. The staining solution has the following com- 
position : — 

Concentrated alcoholic solution of methylene blue, . i part, 
Potash solution ( I in 10,000), . i " 

The potash solution must be added immediately before 
use. 

Cover-glasses are immersed in the mixture for five 
minutes, and afterwards washed in water containing two 
or three drops of acetic acid. Next the preparations are 
transferred to another mixture containing two drops of 
concentrated sulphurous acid, and one of a 5 per cent, 
solution of oxalic acid in 10 c.c. of water. The alkaline 
staining fluid is thus decomposed, and the color removed 
from everything except the bacilli. 

Although not occurring in discharges, two other parasites 



EXAMINATION OF DISCHARGES. 



307 



may be mentioned here, both being more common in the 
flesh of cattle than in man, but, nevertheless, it is import- 
ant to be able to recognize them. 

Trichina spiralis (Fig. 62). — This nematode has 



Fig. 62. 




Trichina spiralis in Muscle (^Farkes). 

already been referred to on p. 247. In its characteristic 
form it is best seen in sections of muscle. It then appears 
coiled up in a lemon-shaped capsule, lying parallel to the 
muscular fibres, and surrounded at each end by a col- 




Head of Cysticercus cellulosae [Parkes). 

lection of fat globules. In some specimens the worm can 
hardly be distinguished, looking like a black mass, and 
the capsule will then be found to be calcified. 

Cysticercus cellulosae. — This has in rare cases 



3o8 MEDICAL MICROSCOPY. 

been found in man. A case is recorded by Dr. Betham 
Robinson in the ^' Transactions of the Pathological 
Society," 1889, p. :^8S, as occurring in the trapezius 
muscle of a child, aged four. Most cases that have been 
reported have been in the eye and brain, where its 
presence is most conspicuous, but in animals it is found 
chiefly in the muscles. It occurs as a small round body 
visible to the naked eye, being sometimes as large as 
i^ inch in diameter. Under the microscope the head 
(Fig. 61,) will be seen to have four suckers and a circle of 
booklets. 

Contents of Cysts. 

For the purpose of diagnosis the contents of cysts are 
usually obtained by means of an exploring needle, but 
if possible it is better to procure a larger quantity by 
aspiration. The fluid is allowed to stand for some 
hours, and then some of the deposit is removed by a 
pipette, and examined by the microscope in the usual 
way. 

Hydatid Cysts. — The fluid obtained from these cysts 
is clear and limpid, with a specific gravity of about 1008. 
It contains very little albumin, and abundance of inor- 
ganic salts. - 

The deposit is characteristic ; it contains the immature 
form of the Taenia echinococcus, a parasite which inhabits 
the intestine of the dog. The ova are discharged with 
the faeces of the host, and in some way, probably through 
drinking water, find their way into the human stomach, 
and thence into some other organ, generally the liver. 
There they develop and form minute round bodies 
known as scolices (Fig. 64). In the sediment these 
can hardly be seen with the naked eye. They are 
provided with two circles of booklets and four suckers. 
Each scolex is oval in form, transparent, and often studded 



EXAMINATION OF DISCHARGES. 



309 



with granules, which are frequently stained yellow by the 
bile. In addition free booklets will also be seen scattered 
through the specimen. In shape these resemble tiger's 
claws. 

In most specimens of this parasite, the original cyst 
formed from the ovum usually develops another within 
itself, known as the ^Maughter-cyst,*' and these again 
develop still more, *^ granddaughter-cysts." In this way 
the echinococcus becomes filled with a number of small 
vesicles of various sizes, which may amount to thousands. 

Fig. 64. 





Hydatid Cysts. 



This illustrates the difference between a '^cysticercus '' 
and an ^^echinococcus;" whereas the former gives rise 
to only one scolex, and can ultimately form only a single 
tapeworm, the latter may develop thousands of both. 

Occasionally the parasites cannot be found, but only 
portions yf the cyst wall (Fig. 65) or ^^cuticula." This 
is made up of a number of concentric layers, composed 
of a chitinous material which has a very characteristic ap- 
pearance, and when once seen cannot be mistaken. 

These cysts occasionally suppurate, and the booklets 



3IO 



MEDICAL MICROSCOPY. 



then are generally the only recognizable feature that 
remains. 

Ovarian Cysts. — The fluid contents of ovarian cysts 
vary considerably in character. The fluid obtained may 
be either perfectly limpid or of a gelatinous consistence. 
The deposit, as a rule, is copious. The cells which are 
formed are but seldom well preserved, fatty degeneration 

Fig. 65. 




Wall of Hydatid Cyst {Boyce). 

being so far advanced that they are scarcely recognizable ; 
free nuclei are abundant. Epithelial cells may be either 
squamous, columnar, or ciliated. Colloid concretions 
which are probably derived from epithelium, are an in- 
variable component of '^colloid cysts." Red and white 
blood corpuscles occur in varying numbers, and cholesterin 
crystals are also generally present. 



EXAMINATION OF DISCHARGES. 3II 

Dermoid Cysts. — The fluid in these cysts is thick 
and brownish in color. The deposit is very copious, con- 
taining, in addition to epithelium, hairs and various forms 
of crystals, especially fat, cholesterin, and haematoidin. 

Hydronephrosis. — The diagnosis of cystic kidney 
can best be assured by chemical tests, namely, those for 
the detection of uric acid and urea. 

The deposit, though usually slight, is of importance, 
and must be carefully collected in a conical glass. On 
microscopic examination epithelium from the renal tu- 
bules must be sought for, and, if found, a diagnosis of 
cystic kidney may safely be made. 

Spermatocele. — The nature of such cysts is at once 
determined by the presence of spermatozoa (Fig. 33, 
p. 188), and occasionally crystals closely resembling the 
Charcot-Leyden crystals. 



CHAPTER XX. 

DISCHARGES FROM THE GENITAL ORGANS. 

A microscopical examination of discharges from the 
genital organs often yields important information, espe- 
cially from a medico-legal point of view. A medical man 
may be called upon to decide as to whether certain stains 
on linen are spermatic or not, or whether a piece of mem- 
brane is derived from the decidua of an early abortion. In 
this chapter a brief description will be given of the most 
important objects seen under the microscope in the exam- 
ination of such discharges. 

For full details and the conclusions to be derived from 
them the reader is referred to treatises on Medical Juris- 
prudence and Obstetrics, although I have been unable to 
find any accurate descriptions of the various forms of 
membrane which may be discharged from the female 
genital organs. - 

Discharges from the Male Genital Organs. 

Seminal Fluid. — Semen, when fresh, is of a viscid, 
honey-like consistence, somewhat opaque, of slightly alka- 
line reaction, and possesses a faint characteristic odor. As 
it cools it becomes temporarily gelatinous, and afterward 
changes into a thick liquid. Its normal color is white, but 
under various conditions this may be altered. 

If mixed with blood it may appear red, brown, or yellow. 
The blood may be derived from the prostatic urethra,, in 
which case spots of pure blood are usually noticed in 

312 



DISCHARGES FROM GENITAL ORGANS. 



3^3 



addition to the brownish color. If the hemorrhage come 
from the vesicul?e seminales, the spots are all equally 
colored, denoting an intimate mixture of blood and semen 
(Ultzmann, ''Sterility and Impotence," p. 15). 

A yellow color is usually due to the admixture of pus, 
and the stains on linen are then greenish or yellowish. 

Occasionally indigo is present, and a violet or claret 
tinge is produced. 

Fig. 66. 




Normal Semen (Ultzmann). 
a. Spermatozoa, b. Seminal cells, c. Epithelium, d. Seminal granules. 

Under the microscope the most prominent objects are the 
spermatozoa (Fig. (^6, a). In normal conditions these 
are present in large numbers. In fresh semen they are seen 
in active movement. They are about fifty p. in length. 
The *' head " is pear-shaped, and 4.5 /x long. The tail is 
tapering ; if a little water is added it will be seen to roll up 
and form a loop. If the organisms are dead, the tail will 
be seen to be spirally coiled or bent at an angle. Their 
27 



314 MEDICAL MICROSCOPY. 

characteristics are very well displayed by the addition of a 
drop of iodine solution. 

A question of sterility may be raised; when the micro- 
scope may be of great value. 

Distinction must be drawn between Aspermatism and 
Azoospermism. 

By aspermatism is meant a condition in which there 
is inability to ejaculate semen. It may be permanent or 
temporary, congenital or acquired. 

In azoospermism, although fluid may be ejected, no 
spermatozoa can be found, and the individual in question 
therefore is sterile. This condition also maybe permanent 
or temporary, congenital or acquired. 

The spermatozoa may be greatly diminished in number 
without being quite absent; this is known as Olizoosperm- 
ism. Such a condition is common in advanced age, or 
may be produced by disease, such as gonorrhoea. 

As long as the spermatozoa can be seen to exhibit active 
movements, the patient cannot be said to be absolutely 
sterile, although impregnation is improbable. 

In addition to spermatozoa, certain other bodies are seen 
under the microscope in semen. 

Seminal Cells (Fig. 66, F), — These are circular in 
shape, finely granular, and represent the spermatozoa 
in an embryonic state ; they usually exhibit one or more 
nuclei. 

Epithelium (Fig. 66, c), — These are of the squamous 
or columnar varieties. 

Masses of amyloid substance, which are finely gran- 
ular within, stratified, and enclose a central nucleus. 
These are derived from the prostatic secretion (von 
Jaksch). 

Seminal Granules (Fig. 66, d). — These form a fine 
molecular debris. They are often found attached to the 



DISCHARGES FROM GENITAL ORGANS. 315 

seminal casts, which have been already described on page 
188. 

Spermatic Crystals. — In general form these bodies 
closely resemble the Charcot-Leyden crystals (see page 
274). Many authors consider them to be chemically the 
same. Fiirbinger asserts that they are derived entirely 
from the prostatic secretion. In normal semen they are 
not deposited for several hours and are seen to be most 
numerous and most perfectly formed in cases of azoo- 
spermism. 

Spermatic Stains. — When semen has dried on linen 
or cotton, the fibre of the stuff is stiffened and the stain 
has a slightly translucent appearance. 

In order to demonstrate the nature of the stain, small 
pieces of the linen should be carefully separated and placed 
in a watch-glass with a few drops of water, just sufficient 
to soak the fibre thoroughly. 

The watch-glass should be covered to keep out dust and 
prevent evaporation, and set aside for an hour. Afterward 
some of the fibres are removed and gently pressed on clean 
glass slides. A slightly turbid fluid will exude, which can 
be rendered clearer, without injury to the specimens, by 
the addition of a little dilute acetic acid. On application 
of cover-glasses, and examining the preparations under a 
quarter-inch lens, the dead spermatozoa will be found if 
the stain was caused by semen. Observation should at the 
same time be made of accompanying hairs or fibres of 
dress stuff, etc. If too much fluid has been added, the 
spermatozoa may become so transparent as to be almost 
invisible. One or two specimens should, therefore, be 
allowed to dry by evaporation, and a couple of drops of 
iodine solution added to the sediment ; the organisms will 
then be well seen. 

Discharges from the genital organs in cases of gonor- 



3l6 MEDICAL MICROSCOPY. 

rhoea and syphilis may be examined for the charac- 
teristic organisms. The methods for so doing have been 
described on pages 305 and 306. 

Discharges from the Female Genital Organs. 

The vaginal secretion under normal circumstances 
exhibits under the microscope squamous epithelium cells 
and a few large leucocytes. 

When the leucocytes are very numerous, vaginal catarrh 
is probably present, and a few red blood cells will be seen 
mixed with the white corpuscles. 

Cancer Cells. — In cases of cancer affecting the 
vaginal portion of the os uteri, in which there is ulceration, 
large epithelial cells, irregular in shape and containing 
large nuclei, are stated to be found in large numbers and 
to be true ^^ cancer cells," but it is very doubtful, and 
whether they can be distinguished from the epithelial cells 
of the vagina, etc., I am much inclined to agree with those 
who consider '' cancer cells '' to be a myth. 

Parasites : — 

Trichomonas vaginalis. — In chronic catarrh of the 
vagina, in addition to a large quantity of epithelial cells 
and mucus and pus corpuscles, an infusorium is found 
known as the ^* trichomonas vaginalis'' (Fig. 67). It is 
oval in shape, and has a long caudal appendage, three 
flagella, and a lateral row of cilia. In the specimen from 
which the accompanying sketch was taken the cilia could 
not be seen. 

Fungi. — Numerous micrococci are nearly always found 
in the vaginal secretion. They are greatly increased after 
childbirth. 

Tubercle bacilli have occasionally been found. 
Cover-glasses should be prepared and stained in the usual 
way (see p. 39). 



DISCHARGES FROM GENITAL ORGANS. 



317 



Gonococci (see p. 306) are found in patients suffering 
from gonorrhoea. 

In cases of suspected rape, the vaginal mucus should be 
examined for spermatozoa. 

Membranes. — Various forms of membrane may be 
passed from the female genital organs, and the microscope 
may be of great aid in helping to decide the nature of the 
case. 

Mucus coagulated by astringents may simulate a 
membrane. By floating it out in water its true composi- 

FiG. 6^, 



-'"^^w^Si 




Trichomonas vaginalis. 



tion will probably be recognized ; if placed under the 
microscope, a large number of cells about the size of leu- 
cocytes will be seen variously altered in shape and irreg- 
ularly distributed amongst ill-defined fibres. When an 
attempt is made to tease the material out, it breaks up into 
fragments, so that it will be found almost impossible to 
obtain a thin layer, and the component parts do not 
adhere together, as is the case with true membrane. 

Fibrinous Clots. — These may closely resemble the 
menstrual decidua of membranous dysmenorrhoea. On 



3l8 MEDICAL MICROSCOPY. 

floating in water, however, the clot is easily broken up. 
Under the microscope red blood cells are seen in abun- 
dance, together with some fibres, the former constituting 
the chief bulk of the specimen. 

Vaginal Casts. — These are easily recognized with the 
microscope. They consist almost entirely of squamous 
epithelial cells closely packed together, amongst which a 
few leucocytes and red blood cells may be seen. Their 
characteristics are best demonstrated by the addition of a 
couple of drops of a two per cent, solution of methylene 
blue. 

Membranous Dysmenorrhoea. — In this condition, 
during the menstrual period, generally on the second or 
third day, membrane is expelled either in shreds or form- 
ing a more or less complete cast of the uterus. 

Under the microscope, layers of fibrin enclosing red 
blood corpuscles are seen ; one surface is rough and un- 
even, whilst on the other small, round cells embedded in 
a reticular network may be made out, amongst which a 
few tubular channels are noticed ; these are the apertures 
of uterine glands, and are therefore of high diagnostic value. 
The epithelium lining the ducts is rarely complete, but is 
usually more or less destroyed. 

In complete casts the orifices of the Fallopian tubes can 
be demonstrated, and the whole structure of the mucous 
membrane is seen, including enlarged orifices of glands 
and an unusual amount of fibrillar tissue. 

Membranes of Early Abortion. — The recognition 
of an early abortion is often attended with difficulty; when 
the decidua have fully formed it is, of course, easier, but if 
the event occurs within the first few months a microscopic 
examination may often be of great value. Portions of the 
amnion only may be found. It is a tough membrane, per- 
fectly smooth and transparent. The internal surface is 



DISCHARGES FROM GENITAL ORGANS. 319 

smooth and glistening and consists of a layer of flattened 
cells, each containing a large nucleus. This layer rests on 
a stratum of fibrous tissue, which gives the membrane its 
firmness, and is attached to the inner surface of the chorion. 
No vessels, nerves, or lymphatics can be seen in it. As a 
rule, the amnion and chorion are found together. One sur- 
face of the membrane is then smooth — the amnion — and the 
other rough, owing to the presence of the chorionic villi. 

If the membrane be teased out and examined under the 
microscope the following structures can be distinguished, 
although their exact distribution will probably have been 
disturbed. 

1. A large number of small, round cells. 

2. A certain amount of fibrous tissue. 

3. Irregular spaces filled with red blood corpuscles, 
many of them looking like large blood vessels. 

4. Flattened cells with large nuclei ; these belong to the 
amnion. 

5. Whorl-like collections of young connective tissue 
cells. These formations are circumscribed, and consist of 
delicate branching fibres with nuclei. They are the chori- 
onic villi. Their appearance is very characteristic, and 
when once seen can hardly be mistaken. I have not 
found anything precisely similar to them in any other 
structure, and I think one would be safe in diagnosing 
foetal membranes when they are present. 

6. A few cubical cells with large nuclei, the so-called 
^* lentil-shaped " cells (Virchow). These are derived from 
the decidua. 

If the decidua are also seen the arrangement of the 
layers is usually more distinct and have the following 
arrangement : — 

On one surface are the flattened cells of the amnion, 
and beneath is the young connective tissue of the chorion. 



320 MEDICAL MICROSCOPY. 

Next comes a thick layer of the " lentil-shaped" cells, 
those nearest the foetal membranes being closer together 
and smaller than those nearer the uterine surface. Then 
a certain amount of glandular tissue will probably be seen, 
the epithelium lining the ducts being often perfectly intact, 
and, finally, some muscular bundles detached from the 
uterus, the characteristic portions being the 'Mentil- 
shaped *^ cells just alluded to. 



CHAPTER XXL 
EXAMINATION OF THE BLOOD. 

A microscopical examination of the blood is usually 
undertaken for the purpose of ascertaining the relative 
proportion in number of the red and white corpuscles to 
the normal and to one another. In addition, certain 
changes in form of the red corpuscles are of importance, 
and the amount of coloring matter is also usually esti- 
mated. More rarely, various micro-organisms are sought 
for. 

In normal blood the proportion of white to red cells is 
about I to 300, this varying considerably within physio- 
logical limits ; thus, the white cells are greatly increased 
in number after a meal, when the proportion may rise to 
I to 150, afterward falling to i to 600. 

The red corpuscles are -32V-0 inch (7.5 ti) in diameter, 
and Y2V0 ^^^^ (2 /^') in thickness. When seen sideways 
they are bi-concave or dumb-bell shaped and of a pale buff 
color, which appears of a reddish tint when a number of 
corpuscles are collected together. They have a tendency 
to run together, forming rolls or rouleaux. They are non- 
nucleated. 

When the circumference of a corpuscle is brought 
sharply into focus, a darker area is seen in its centre, but if 
the lens is brought nearer the corpuscle by means of the 
fine adjustment, the darker centre is replaced by a lighter 
area, while the ring becomes darker ] this being due to the 
fact that these bodies are bi-concave, circular discs. 
28 321 



322 MEDICAL MICROSCOPY. 

The white corpuscles are usually spherical in form 
and faintly granular. If the blood is examined fresh on 
a warm stage, amoeboid movements may be noticed. 
Their average size is about ^5^00 inch, or 10 /jl in diameter. 
One or more nuclei are present, but are usually not 
visible unless a little weak acetic acid is run under the 
cover-glass. 

A third element will sometimes be observed, namely, 
blood-plates. These were first noticed by Bizzozero. 
To demonstrate them the blood must be *' fixed " by the 
addition of some preservative fluid, such as Hayem's. 
This has the following composition : — 

Chloride of sodium, I part, 

Sulphate of sodium, 5 parts, 

Corrosive sublimate, 5 part, 

Distilled water, 200 parts. 

A couple of drops of this fluid is added to a drop of 
the blood obtained from the finger and placed on a glass 
slide. A cover-glass is applied, and the specimen ex- 
amined by the highest obtainable power (preferably a y^- 
oil immersion) and a small diaphragm. The plates will 
then be seen as slightly oval or rounded granules about 
one-third the size of the ordinary red corpuscles. 

Methods of Examination. — For the histological 
examination of the blood it is sufficient to tie a handker- 
chief tightly around one finger, so as to compress as 
much blood as possible into the tip, then prick it with a 
sharp needle, and remove the drop which exudes on a 
clean cover-glass. This should be placed on a glass slide 
and its edges surrounded with a rim of oil or Canada bal- 
sam, in order to prevent evaporation. 

Two processes of the utmost importance in the ex- 
amination of the blood will now be described, /V ^., 
^^ counting the corpuscles" and the ^'estimation of 



EXAMINATION OF BLOOD. 



323 



hsemoglobin." The latter does not strictly come under 
the head of Medical Microscopy, but a chapter on ex- 
amination of the blood for clinical purposes can hardly be 
complete without it. 

Counting the Corpuscles. 
This is usually accomplished by means of Dr. Gowers' 
hsemocytometer, Fig. 6S (made by Havvksley, Oxford 

Fig. 68. 




Gowers' Hoemocytometer. 

A. Pipette for measuring the diluting solution. B. Capillary tube for measuring 
the blood. C. Cell with divisions on the floor. D. Vessel in which the dilution 
is made. E. Spud for mixing the blood and solution. F. Guarded spear- 
pointed needle. 

Street), which consists of (i) a pipette graduated to 995 
cubic millimetres for measuring the diluting solution ; 
(2) a capillary tube for measuring the blood .and capable 



324 MEDICAL MICROSCOPY. 

of holding 5 cubic millimetres ; (3) a small glass jar and 
stirrer for making the dilution ; and (4) a glass slide on 
which is arranged a cell . 2 millimetre deep, and ruled at 
the bottom in squares, each .1 millimetre in length and 
breadth. The slide is attached to a metal plate furnished 
with two springs for maintaining the cover-glass in position. 
The diluting solution recommended by Dr. Gowers is 
as follows : — 

Sulphate of soda, 104 grains, 

Acetic acid, i drachm, 

Distilled water, '. . . 6 ounces. 

The instrument is used in the following manner: — 
995 cubic millimetres of the saline solution are mea- 
sured off in the pipette and placed in the mixing glass. 
A drop of blood is obtained from the patient's finger in 
the way described above, and drawn up by the capillary 
tube to the level of the 5 cubic millimetre mark, and then 
thoroughly mixed with the saline solution in the glass. 
One caution must here be given, and that is that if the 
drop of blood does not readily exude, too much pressure 
must not be applied, or otherwise too great a proportion 
of serum will be obtained, and thus an incorrect calcula- 
tion will result. 

The diluted blood is then thoroughly stirred with the 
small glass rod ; one drop is transferred to the floor of the 
cell and another to a cover-glass. The two drops are then 
approximated, which insures a regular layer extending 
throughout the depths of the cell, and the springs are 
arranged so as to keep the cover-glass in position. It is a 
good plan, before placing the blood in the cell, to rub the 
ruled portion of the cell over with a soft black-lead pencil, 
afterward removing the loose dust. This renders the 
squares distinct, otherwise, especially in a new glass, they 
are often barely visible (Wynter). 



EXAMINATION OF BLOOD. 325 

The slide is now examined with a quarter-inch lens, and 
the number of corpuscles counted in ten squares. In 
order to explain the computation, we quote Dr. Gowers' 
own words, in his article in Quain's ^* Dictionary of Medi- 
cine : " — 

*^ The dilution of 5 c.mm. of blood in 955 c.mm. of 
solution is i in 200; each square contains the corpuscles 
from a volume of dilution .2 mm. in one and .1 mm. in 
each of the other dimensions — that is, 2 cubic .1 mm., or 
the .002 part of a cubic mm. But the dilution being i in 
200, this volume of dilution contains just .00001 cm. of 
blood. The number of corpuscles in a square multiplied 
by 100,000 is thus the number in a cubic millimetre of 
blood — the common mode of statement. In order to limit 
error, the total number of corpuscles in ten squares should 
be counted, and this number multiplied by 10,000 is the 
number per cubic millimetre." 

The average number in normal blood is about 5,000,- 
000, which corresponds to about fifty in a square; from 
this it is evident that normal blood will contain 100 cor- 
puscles in two squares, and that, therefore, the number of 
corpuscles of any blood in two squares of the haemacyto- 
meter represents the percentage of corpuscles compared 
with normal blood. 

In counting the white corpuscles, if they are not in con- 
siderable excess, it is most convenient first to ascertain the 
number of red corpuscles per square and note how many 
squares are contained in the field of the microscope. If, 
then, the focus is raised, so that the corpuscles gradually 
become indistinct, the white ones, from their higher 
refracting power, will appear like bright points, and the 
number in a series of fields can easily be counted. For 
example, the number of red corpuscles per square has been 
found to be 40, and the field contains fifteen squares, 



326 



MEDICAL MICROSCOPY. 



Fig. 69. 



llOl 



0'$ 



Pipette (Thoma-. 
Zeiss Hsemocyto- 
meter). 



that is, 600 corpuscles per field. Ten 
fields contain fifteen white corpuscles; 
the proportion of white to red will, 
600 X 10 



therefore, be 
400. 



I to 



15 



I to 



Another excellent instrument, though 
not so well known in this country, is the 
Thoma-Zeiss hcemocytometer (may be 
procured from Baker, 243 High Hol- 
born). Dr. Archibald Garrod has had 
considerable experience in the use of 
this apparatus, and I have to offer him 
my best thanks for kindly writing for 
me a description of the haemocytometer 
and the mode of using it. 

The dilution and mixing of the blood 
with saline solution is carried out in a 
pipette (Fig. 69) made after the pattern 
of Potain's melangeur. The capillary 
bore of this pipette is at one point ex- 
panded into a bulb in which lies a small 
glass ball. The lower portion of the 
tube is graduated, and into it are drawn 
five or ten cubic millimetres of blood ; 
saline solution (of the composition given 
above) is then drawn in until the bulb, 
and a very small portion of the capillary 
tube beyond, are filled with the mixed 
blood and solution. According to the 
amount of blood taken, the dilution will 
be 200 or 100 times. A gentle rotary 
motion is then imparted to the pipette, 
which, by causing the glass ball to move 



EXAMINATION OF BLOOD. 



327 



within the bulb, brings about a complete mixture of its con- 
tents. A small portion of the liquid is then expelled, so as 
to clear out the unmixed saline solution from the capillary 
tube, and afterward a drop of the diluted blood is placed 
upon the counting slide (Fig. 70). The centre of this slide 
is formed into a circular cell by a rim of glass, and in the 

Fig. 70. 




Counting Slide, Thoma-Zeiss Apparatus. 

middle of the cell so formed is a small platform not quite 
so thick as the surrounding rim (Fig. 71). When the 
cover-glass is placed over the cell, a space of .01 millimetre 
is left between it and the central platform, whilst the small 
trough between the platform and the rim serves as a recep- 
tacle for any overflow of fluid. 

Fig. 71. 



Cross Section of Thoma-Zeiss Apparatus. 

When the cover glass is accurately applied, Newtonian 
colors will be produced ^vhere it is in contact w^ith the rim 
of the cell. When these colors cannot be obtained, it 
must be concluded that some dust is lying between the two 



328 



MEDICAL MICROSCOPY. 



surfaces of glass, which must then be separated and cleansed, 
and a fresh drop of liquid from the pipette placed in the 
cell. The preparation must also be rejected if any of the 
liquid makes its way between the cover-glass and the ex- 
ternal rim. 

Upon the centre of the platform is a square divided into 
400 lesser squares^ each ^i^ square millimetre in area and 
2V nim. in diameter. Since the depth of cell is .01 c.mm. 
the space over each square is 40V0 c.mm. The contents of 
16 such squares gives the percentage of corpuscles. Six- 

FiG. 72. 



















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-e 


% 




' 








°' 


,%' 


° I ° 


>° 




,' 


°o = 


a-'o 






V,- 


» 




** 0° 


9 
<3 6 


' « 
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"o'e 


c°6 


; 









«> 


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°c; 


^° 


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°o 


o] 









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"0 


o'>\ 


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>°° 




1° 


** 6 






a e 


°o 


y 





*° 






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}° 


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00 


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= 0° 






=. » 


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n-.ei 





9° 




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i 





Counting Scale, Thoma-Zeiss Apparatus, 



teen sets of sixteen squares are marked off by triple lines 
(Fig. 72), and if the whole of these are counted, or 256 
squares in all, the area included in the counting equals 
that of 32 squares of Gowers' haemocytometer. 

In order to estimate the number of red corpuscles per 
cubic millimetre of undiluted blood, if after counting the 
corpuscles in a certain number of squares, m be found to 
be the average number of corpuscles per square of 4^0 niil- 
limetre area, this number would have to be multiplied by 
4000 X 100 or 200 (according to the dilution taken) to 



EXAMINATION OF BLOOD. 329 

give the result sought for. Or the calculation may 
be performed thus : 4000 x 200 (or ioo )_>^^ _ ^^^ where x = 

total number of corpuscles counted, y = the number of 
squares, and the result {n) will be the number of corpuscles 
in a cubic millimetre of undiluted blood. 

Dr. Garrod says : '^ I always count the squares in sets 
of 16, counting successive rows of four squares, including 
in my counting those corpuscles which touch the upper and 
left-hand borders of the square, either from the outer or 
inner side, and excluding those which touch the lower and 
right-hand borders." 

The small size of the squares of this instrument very 
greatly facilitates the process of counting ] which may 
also be interrupted, if necessary, and continued later on 
from the point at which it was stopped, as a note can be 
made of the number and row of the square last counted. 

One of the drawbacks to the instrument is the trouble 
required for the cleaning of the pipette, which should be 
washed out several times with water and then dried. The 
drying is effected by means of alcohol, and after the ex- 
pulsion of the spirit a current of dry air should be sent 
through the pipette until the little glass ball moves freely 
about in the bulb, without adhering to the sides. The 
pipette should also be washed out occasionally with ether, 
and at intervals with caustic potash. 

For the counting of the white corpuscles a second pipette 
is provided, of similar form, but giving a dilution of 10 or 
20 times. The liquid employed is a ^ per cent, solution 
glacial acetic acid, which destroys the red corpuscles and 
at the same time renders the white more clearly visible. A 
drop of the mixed blood and acid is placed in the cell and 
the corpuscles are counted by fields of the microscope, the 
area of which is determined in the following manner: — 



330 MEDICAL MICROSCOPY. 

The diameter of the field is ascertained by means of 
divisions on the floor of the cell, each of which equals 
-^ millimetre. If the field has a chamber of ten divisions, 
the radius will be \ millimetre, and the area tt (i)2 square 
millimetre, and since the depth of the cell is .01 milli- 
metre, the cubic contents of the field will be o.i X ^ (i)^ 
cubic millimetre (jz = 3. 141 6). The number of white cor- 
puscles may be then calculated by means of the following 
formula : — 

D X N 

FX C 

when D = the dilution, /. e., 10 or 20, 

N = number of corpuscles counted, 
F = number of fields counted, 
C = cubic contents of a field. 
In practice the calculation may be greatly simplified by 

always counting the same number of fields {e.g., 50) and 

by always using the same sized field and the same dilution 

of the blood. 

Estimation of the Amount of Haemoglobin in the 

Blood. 

Various modes of estimating the richness of the blood 
in haemoglobin have been introduced. It may be ac- 
complished by the amount of dilution necessary to obscure 
a certain absorption band in the spectrum, or by the col- 
ored discs invented by Hayem. The two most practical 
methods for clinical purposes are those of Dr. Gowers 
and von Fleischl. 

Gowers' Hsemoglobinometer (Fig. 73). — This ap- 
paratus consists of two small tubes of exactly equal dia- 
meter, which when in use are placed in a stand made to 
receive them. One tube contains a preparation of carmine 



EXAMINATION OF BLOOD. 



33^ 



and glycerine jelly colored to represent a solution of i part 
of healthy blood in loo of water. 

The other tube is graduated, each division being equal 
to the volume of blood taken (20 cubic millimetres). So 
that 100 divisions equal 100 times the volume of blood. 
A capillary pipette is also supplied capable of holding 20 

Fig. 73. 






Govvers' Hoemoglobinometer. 

A. Bottle for holding the diluting solution. B. Capillary pipette for measuring the 
blood. C. Graduated tube for measuring the amount of haemoglobin. D. 
Standard tint of normal blood. E. Support for D and C. F. Puncturing 
needle (Haivksley). 

cubic millimetres. This is filled with blood which is 
blown into the graduated tube in which a few drops of dis- 
tilled water have previously been placed. The blood and 
the water are then thoroughly mixed. Distilled water is 
now added drop by drop from the pipette until the tint of 
the mixture corresponds with that of the standard. 

The height of the diluted fluid is then read off, the 



332 



MEDICAL MICROSCOPY. 



division which is reached representing the percentage 
amount of haemoglobin. 

For example, a specimen of blood is diluted until the 
mixed fluids reach the 55 mark on the tube, when the tint 
of the standard is found to be attained. The blood ex- 
amined, therefore, contains 55 per cent, of the normal 
quantity of haemoglobin. 




Von Fleischl's Hsemometer (for explanation of lettering see text). 



Von Fleischl's Haemometer (Fig. 74). — For an 
account of this excellent piece of apparatus, I am once 
more indebted to Dr. Archibald Garrod, who describes it 
and the means of using it in the following words : — 

^^In this instrument the standard of comparison is an 
elongated wedge of tinted glass (K), which is so mounted 
that it can be moved backward and forward beneath a 



EXAMINATION OF BLOOD. ^7,^ 

small stage by turning a milled wheel (T). In the stage 
is a circular opening through which the light of a lamp 
is reflected upward from a disc of plaster-of-Paris (S) 
mounted like the mirror of a microscope. The wedge of 
glass underlies one-half of this opening. The mixture of 
blood and water to be examined is placed in one division 
of a cylindrical vessel (G), which has a glass bottom, and 
is divided in half by a vertical septum (a). This vessel 
fits accurately over the circular opening in the stage, and 
must be so adjusted that the septum coincides with the edge 
of the tinted prism. On looking down upon the instru- 
ment, one-half of the illuminated circle will then receive 
its tint from the prism, the other half from the mixture 
contained in the vessel. 

'^The blood is received from the ear or finger into a 
minute glass tube, into which it is drawn by capillary at- 
traction. This tube is held by a small metal handle, which 
afterward serves as a convenient stirring rod. The tube 
being accurately filled, and any blood which may have 
adhered to its outer surface having been carefully removed, 
its contents are im7nediately washed out into the mixing 
vessel by means of a small pipette filled with water. 
Water is then added with careful stirring, until a plane 
surface is obtained, level with the top of the cylindrical 
vessel, and showing neither a concave nor a convex menis- 
cus. If any clotting has occurred in the tube, a fresh 
specimen should be taken, but after a little practice this 
accident will seldom occur. 

'' The other half of the vessel, which lies over the tinted 
prism, must then be filled in the same manner, with pure 
water. The comparison of the tints is facilitated by plac- 
ing a cardboard tube, blackened internally, upon the stage 
of the instrument, so as to enclose the mixing vessel. 

*' The prism is then swept backward and forward until 



334 MEDICAL MICROSCOPY. 

an accurate match is obtained, when the percentage of 
haemoglobin may be read off upon a scale (P) which is 
visible through a second small opening (M) in the stage. 
It is well to repeat the comparison several times, until the 
successive readings exhibit a close agreement. 

^* The estimate should be arrived at as quickly as pos- 
sible, because the eye soon becomes tired, rendering an 
accurate comparison almost impossible. In case of doubt 
the eye should be rested, and a fresh determination made 
after an interval. 

^^ The comparison requires a darkened room and an 
artificial light, since by daylight the tint of the prism is 
entirely unlike that of the diluted blood. 

'' The standard of haemoglobin is somewhat higher than 
that of Gowers' haemoglobinometer, and I have not yet 
found the blood of any Londoner to attain to the loo 
per cent. 

^' I have made repeated observations in quick succession, 
taking a fresh specimen of the blood of the same individ- 
uals, and have obtained results not differing by more than 
I per cent., but it should be mentioned that the scale is 
only divided into tens and fives, although I believe that 
the delicacy of the instrument would admit of finer gradu- 
ation. As it is, the units of percentage must be estimated 
by the observer." 

Having described the methods of estimating the number 
of the corpuscles, we must now proceed to consider what 
results may be arrived at from this computation. The red 
corpuscles may be decreased, this condition being known 
as oligocythsemia. As before stated, the normal number 
of red corpuscles in the blood is 5,000,000 in a man and 
4,500,000 in a woman to the cubic millimetre of blood. 
In various forms of disease this number may be diminished. 



EXAMINATION OF BLOOD. 335 

the number falling to 2,000,000, or even as low as 360,000 
per cubic millimetre (v. Jaksch). The lowest number I 
have ever found was 450,000 ; this occurred in a case of 
pernicious anaemia. 

Temporarily this decrease may be due to hemorrhage. 
As a permanent condition it is found in those diseases which 
are attended with deficient production of the corpuscles. 

The quantity of haemoglobin is roughly proportionate to 
the number of corpuscles, but the individual corpuscles 
often contain less haemoglobin than normal. The loss 
may be so great that the proportion of haemoglobin falls 
to 25 per cent, of the normal, as in some cases of chlorosis. 
Hayem drew attention to the fact that in pernicious anae- 
mia the number of red cells in the blood is inversely pro- 
portional to the quantity of haemoglobin which they con- 
tain. In the case just referred to, the blood, tested by v. 
FleischFs haemometer, only yielded ten per cent, of haemo- 
globin. 

Polycythaemia. — Increase in number of the red cor- 
puscles is never great, being very transitory and within 
physiological limits; thus it is found after meals and in the 
newly born. In plethoric conditions also the number is 
above the average, and in the algid stage of cholera the 
red corpuscles are relatively in excess. 

Leucocythsemia. — By this is meant a condition in 
which there is a considerable and permanent increase in 
the number of the white red corpuscles ; it is also known 
as leicccemia. 

Microscopical examination sometimes shows an enor- 
mous increase in the number of the white corpuscles. 
Virchow has found them as high as in the proportion of 
2 : 3 of red corpuscles. In this condition the red cor- 
puscles are also diminished, being generally about two or 
three millions to the cubic millimetre of blood. 



336 MEDICAL MICROSCOPY. 

Von Jaksch {loc. cit,, page 20) has made some remark- 
able observations as regards the character of the leucocytes. 
It is usual to distinguish leucaemia as splenic, lymphatic, 
and myelogenic, according to the anatomical seat and 
clinical symptoms of the disease. When large and small 
leucocytes, the latter preponderating, occur, the leucaemia 
is of a lymphatico-splenic character. When the larger cells 
alone are found, v. Jaksch considers that the disease is 
almost entirely splenic. If the corpuscles are of a transi- 
tional form, nucleated red cells being also present, the 
bone-marrow is probably the seat of serious changes. 

Although the more pronounced forms of leucocy- 
thaemia are recognized without difficulty, it is not so easy 
to detect an early stage of this condition. 

Prof. Ehrlich has pointed out how this may be ac- 
complished, by taking advantage of the selective pro- 
perties of the ^^ granules" present in the protoplasm of 
the leucocytes toward eosin. 

Preparations are made as follows : Two films of 
blood are procured by pressing a drop of blood between 
two cover-glasses, and then sliding them apart. They 
are next dried in an exsiccator and heated upon copper 
foil for a considerable time (ten to twelve hours) at 120° 
to 130° C. A more simple expedient may be adopted 
by placing the glasses, as soon as the blood has partially 
dried, in absolute alcohol for a couple of hours, and then 
driving off the spirit by heat. A drop of concentrated 
eosin-glycerine solution is then added to each cover- 
glass, the excess of stain is washed off with water, and the 
glasses are finally dried and mounted in balsam. 

Ehrlich found that the granules presented consider- 
able differences in their staining properties. He dis- 
tinguished five several varieties, classifying them as a- to £- 
granules. 



EXAMINATION OF BLOOD. ' 337 

When the disease is acute, and the number of leuco- 
cytes only slightly diminished, the mono- and poly- 
nuclear forms (£-granules) are alone increased, whilst 
the a-granules (*' eosinophile ") are decreased. When 
the condition is a more chronic one, exactly the reverse 
condition of affairs results, the a-granules being increased 
and the e-granules diminished. 

Various changes in the red corpuscles are noted in 
leucocythaemia, but these will be described separately. 

Leucocytosis. — This term is applied to the condi- 
tion in which the white corpuscles are temporarily in- 
creased. This occurs physiologically after a meal, the 
proportion of white rising as high as i to loo red cor- 
puscles. According to Virchow, leucocytosis accom- 
panies every case of lymphatic excitement, such as 
inflammation, and tubercular, scrofulous, or cancerous 
enlargements, or swelling of the glands, and allied 
structures in Peyer's patches, solitary follicles, the spleen, 
and the tonsils. I have found an increase of white cells 
in cases of cancer, when the cachexia has been very 
profound. 

This condition differs from leucocythaemia then in its 
transitory character, by the absence of deficiency of the 
red corpuscles, and by the degree in which the white cor- 
puscles are diminished. 

Von Jaksch and Tumas maintain that leucocytosis occurs 
regularly in croupous pneumonia. 

Microcythsemia. — Various observers have from time 
to time described bodies in the blood which appear like 
small red corpuscles. Manassein ('^ Centralblatt," 1871) 
produced changes in the diameter of the red blood cells 
by introducing chemical substances into the circulation. 
He noticed a diminution in size of the corpuscles in cases 
of hyperpyrexia, in septic poisoning, and when the oxida- 
29 



338 MEDICAL MICROSCOPY. 

tion of the blood was interfered with, as during the in- 
halation of carbonic acid. He found the diameter of the 
corpuscles increased by the action of alcohol and quinine, 
also under the influence of cold, in cases where an excess 
of oxygen had produced an acute anaemia. 

The bodies known as '' microcytes" were first described 
by Vaulair and Masius (^^ Bulletin de I'Academie Royale 
de Med. de Belgique/' 1871, p. 515). They are spherical 
in form, rather redder than the ordinary corpuscles, and 
have a diameter of 2 or 3 /x. They do not run into rou- 
leaux. Some authors (Gram, Hayem) consider that they 
are caused by the fluids added to the blood for diluting 
purposes, but they are seen when no such agents have been 
used, and when the pure blood has been examined directly. 
Portions of coloring matter may be removed by the said 
agents and simulate closely the microcytes, but they will 
be found not to possess such clear outlines, and, as a rule, 
only occur sparsely, whilst the microcytes are usually 
numerous. 

They are found in various conditions, such as infectious 
diseases, pronounced anaemia, and burns. Vaulair and 
Masius met with them in pathological states in which there 
was a diminished activity of the liver, with an increased 
activity on the part of the spleen. I have also observed a 
similar case. In pernicious anaemia they are very abundant ; 
Eichorst goes so far as to consider them pathognomonic 
of this disease. As regards their nature, some authorities 
believe that they are caused by the rapid and uniform ab- 
straction of water from the corpuscles, others that they are 
the final stage of the changes which the red corpuscles 
undergo in the spleen, and that they are destroyed by the 
liver. Their true significance is not yet known, and the 
matter requires further elaboration before any diagnostic 
importance can be attached to them. 



EXAMINATION OF BLOOD. 339 

Poikilocytosis (Fig. 75). — In certain affections many 
of the red corpuscles are found to have undergone remark- 
able changes in shape and size. Some are seen to be much 
larger than usual (megaloblasts), but the majority show 
various departures from the normal spherical form. Some 
are oval, others pear-shaped, others kidney-shaped, whilst 
some exhibit minute processes, like small knobs projecting 
from them. Von Jaksch {Joe. at., p. 24) considers that in 
this condition the red corpuscles are endowed with an 
abnormal power of contractility. 

These appearances were at one time thought to be pecu- 
liar to pernicious anaemia, but have since been observed 

Fig. 75. 



6c7 



" P p O 

Blood in Poikilocytosis. 



in many cases in which the blood is greatly affected, such 
as severe anaemia, of whatever kind, and in chlorosis. 
They have also been recorded as occurring in the blood in 
cancerous cachexia and in myeloid degeneration of the 
viscera. The most pronounced specimen I have ever seen 
was obtained from a patient suffering from gout and pro- 
found anaemia with splenic enlargement. 

To observe these bodies the blood should be examined 
pure, without the addition of diluting fluids. 

Melansemia. — In the blood of patients who have 
suffered for some time from malarial diseases, particles of 
pigment are observed in the blood. These are usually 



340 MEDICAL MICROSCOPY. 

black, more rarely brown or yellow. They are sometimes 
free, and sometimes enclosed in round or oval cells, 
probably leucocytes. 

This appearance is often associated with an enlarged 
and deeply pigmented condition of the spleen ; it is not 
improbable that the pigment particles are produced by 
the malarial fever, and find their way from the spleen into 
the blood. 

Parasites in the Blood. 

Vegetable Parasites. — Several of the most important 
pathogenic bacteria have been found in the blood of 
patients suffering from the particular diseases caused by the 
germs. The number of micro-organisms, however, falls 
far short of that found in animals under similar conditions. 

In order to search for the organisms, the finger of the 
patient should be well scrubbed with soap and water, and 
then washed over with corrosive sublimate of the strength 
of I in I GOG. The tip of the finger is then compressed 
and pricked with a sterilized needle. The drop of blood 
which exudes is taken off with a clean cover-glass and 
immediately covered with another, the two being pressed 
together and then slid apart, so as to obtain two films; 
when dry they are passed three times through the flame, 
and afterward stained in an appropriate manner, according 
to which bacillus is to be sought for. 

Bacillus of Anthrax. — Cover-glasses prepared as 
above should be stained by Gram's method (see p. igi). 
Their appearance does not differ from that described on 
p. 305, as obtained from the local lesion. 

Bacillus of Tubercle. — Cover-glass preparations must 
be stained by Neelsen's method (p. 104). Tubercle bacilli 
are found with difficulty in the blood, and a large number 
of specimens must be obtained. Meisels found them in 



EXAMINATION OF BLOOD. 341 

the blood of a patient suffering from miliary tuberculosis, 
and his discovery has since been confirmed by other 
observers. 

Although I have made several attempts, I have never 
been able to detect the bacilli in the blood of tubercular 
patients. 

Bacillus of Typhoid Fever. — Some observers have 
succeeded in finding bacilli in the blood of patients 
afflicted with enteric fever, which morphologically, in 
their manner of growth on artificial media, and in the 
effect they have when inoculated on animals, are similar to 
those which are found in typhoid stools and in the tissues 
after death (see p. 230). Cover-glass preparations should 
be stained in methylene blue, and washed in water acidu- 
lated with a drop or two of acetic acid. 

Bacillus of Glanders — The pathogenic bacilli of 
glanders, in addition to being found in the farcy buds 
and discharges from ulcers in patients suffering from 
that complaint, have been found in the blood in small 
numbers. They are difficult to stain ; cover-glass prepara- 
tions are best colored by Klihne^s method (see p. 98). 

Bacillus of Tetanus. — Nicolaier and Kitasato were 
the first to describe this organism. The rods are long and 
slender and exhibit terminal spore formation. They have 
only very rarely been found in the blood. 

Spirillum of Relapsing Fever. — The organism 
found in the blood of patients suffering from relapsing 
fever may be demonstrated in a drop of fresh blood placed 
under a cover-glass, or dry preparations may be made in 
the usual way, stained with fuchsine, and washed in water 
containing a few drops of alcohol. 

The spirillum is usually known as the ^^ spirillum Ober- 
meieir," having been originally described by Dr. Obermeier 
of Berlin in 1873. It takes the form of a delicate, 



342 MEDICAL MICROSCOPY. 

homogeneous, spirally-twisted filament, having a length 
of from 2W00 to 3^0 inch (15.40 /x). In fresh blood it 
exhibits active movements, rotating on its long axis, and 
with a lashing movement progressing backward and for- 
ward. 

The ^^spirochaetae," as the spirilla are sometimes termed, 
are only found in the blood during the actual attacks of 
relapsing fever, and cannot be found during the intervals. 
According to Heydenreich, they appear before the febrile 
attack begins, but cease to be discoverable before the com- 
mencement of the crisis. The same observer also finds that 
their numbers vary greatly from day to day. Sometimes, 
after the filaments have been present for two or three days, 
they suddenly disappear, but a few hours later may reappear 
in large numbers. 

They are very sensitive to reagents of all kinds. If the 
blood be examined in the intervals of the disease, peculiar 
refractive bodies resembling diplococci are found ; these 
are especially numerous when the paroxysm commences. 
Von Jaksch (/oc. cit,^ p. 31) considers that when the attack 
begins these bodies grow out, as it were, into short, thick 
rods, from which the spirilla are finally evolved. They 
are therefore probably spores. 

Haematozoa of Malaria. — Lavaran, in 1880, was the 
first to notice the existence of peculiar structures in the 
blood of patients suffering from malaria, and his observa- 
tions have been confirmed by several observers, notably by 
Marchiafava and Celli. 

The different forms met with may be divided into two 
groups — those within the red blood corpuscles and those 
free in the serum. 

Intra- corpuscular Bodies. — There are three varieties of 
these : Firstly, irregular protoplasmic bodies much smaller 
than the corpuscles. They exhibit active amoeboid move- 



EXAMINATION OF BLOOD. 343 

ments. Marchiafava and Celli, who were the first to 
observe them, suggested the name Plasmodium malariae. 
Secondly, minute masses of finely granular or hyaline pro- 
toplasm enclosing granules of pigment. They are usually 
circular in shape, and likewise exhibit amoeboid move- 
ments. The pigment granules also change their shape. 
Thirdly, forms which appear like isolated grains, and in 
addition, large homogeneous bodies surrounded by clear 
spaces. 

Extra- corpuscular Bodies. — These are also of various 
forms. Firstly, what are known as the '' semi-lunar bodies 




Filaria sanguinis hominis (specimen in the urine, Boyce). 

of Laveran.'* These objects are crescent-shaped, either 
rounded or pointed at the extremities ; occasionally, they 
are almost spherical. They are motionless. Pigment gran- 
ules are found in the centre of these bodies, which seem to 
be composed of homogeneous protoplasm. Secondly, pro- 
toplasmic masses, finely granular in appearance. The pig- 
ment is collected into the form of a rosette, and the pro- 
toplasm gradually undergoes segmentation, forming small 
spherical bodies, which are ultimately set free. Thirdly, 
circular, pear-shaped, or ovoid bodies, a little smaller than 



344 MEDICAL MICROSCOPY. 

red blood corpuscles, provided with one or more actively 
mobile, long, lash-like filaments. Fourthly, small, spherical, 
pigmented bodies, about one quarter the size of the red 
blood corpuscles. These exhibit amoeboid movements. 

To demonstrate these bodies fresh blood is examined 
in the usual way. For permanent specimens, dried 
cover-glasses are prepared, and stained by the Gram 
method. 

Klebs and Tommasi-Crudeli found bacilli in the soil 
of the Campagna, which they regard as the specific 
organism of malaria. 

Animal Parasites. 

These are the Filaria sanguinis hominis and the 
Bilharzia hsematobia. 

Filaria sanguinis hominis (Fig. 76). — This para- 
site has already been referred to (see p. 195). The dis- 
covery of the embryos of this nematode worm in the 
blood was first made by Dr. T. R. Lewis in 1872. 
Four years later the parent worm was found by Dr. 
Bancroft, of Brisbane. It has been shown to inhabit 
the lymphatics of the inhabitants of tropical countries. 
It does not necessarily produce disease, and many 
natives (i in 10 in South China) in perfect health were 
found to be affected. Usually the condition is brought 
to notice by the patients becoming afflicted with chy- 
luria or elephantiasis. Dr. Stephen Mackenzie found 
them in the blood of a native of the Upper Congo suf- 
fering from ^* sleeping sickness." The parent worm is 
about 3 to 3^ inches long, and has a diameter of about 
yi-Q inch, and resembles ^^a delicate thread of catgut, 
animated and wriggling" (Manson). The mouth is 
circular, without papillae ; there is a narrow neck, and 
the tail is bluntly pointed. It is with the embryos. 



EXAMINATION OF BLOOD. 345 

however, that we are immediately interested. Specimens 
are obtained by pricking two or three fingers of the 
patient, transferring the drops to a glass, and covering 
with a long cover-glass. 

The embryos are seen as active organisms about gV 
inch in length, and having a diameter ^oo j each is en- 
closed in a delicate sac or sheath which fits it accurately, 
except that a collapsed or unoccupied part is seen project- 
ing at one end. Most observers agree that this sheath is 
the envelope or shell of the ovum, which, as the embryo 
develops, becomes stretched out over the skin. This en- 
veloping membrane is quite structureless, but the con- 
tained parasite is seen under a high power to be trans- 
versely striated and very granular. The embryos only 
occur in the peripheral vessels whilst the patient is asleep, 
generally, therefore, at night. They begin to make their 
appearance at about 6 or 8 p. m. By midnight their num- 
bers reach the maximum. As morning approaches they 
become fewer and fewer, and finally disappear altogether ; 
but that this is not due simply to daylight is shown by 
Dr. Stephen Mackenzie, who caused a patient to be up all 
night and in bed all day, with the result that the filariae 
were only found during the day ; on the other hand, the 
most dense London fog, if the patient was up and about 
during the day, did not tempt the parasites to appear. 

Dr. Mason has twice obtained ova in a much earlier 
stage of development, consisting of oval bodies -^-^ inch 
in length and y-i-Q in breadth. 

Bilharzia hsematobia (Distoma haematobium). — Pa- 
tients who have dwelt in tropical countries and become 
affected with endemic haematuria, have been shown to be 
the hosts of a parasite, to which the name of Bilharzia 
haematobia was given by Dr. Cobbold. The adult para- 
sites (Fig. 77) occupy smooth walled spaces in communi- 
30 



346 MEDICAL MICROSCOPY. 

cation with the veins of the pelvis. This fluke is white in 
color, the male and female being distinct. The former is 
flattened, and about half an inch in length j the hinder 
part is more cylindrical in form, from the edges being 
thinned and folded inward so as to form a groove, in 
which the female is received during sexual congress. The 
female is round and thin, and about ^ inch long. The 
parasite itself is but rarely seen in the blood, but the ova are 
discharged and are often found in the urine (see p. 194). 

Examination of Blood Stains. 
A medical man is occasionally called upon to give a 
decision as to whether a stain on some portion of clothing 

Fig. 77. 




Bilharzia hsematobia, Male and Female; Eggs {vo7t Jaksch). 

is caused by blood. Although in important cases such a 
question is generally referred to an expert, yet all doctors 
ought to be prepared to make such an examination. The 
suspected stain should first be examined by a good lens; 
if caused by blood, it will not be a mere coloring of the 
fibres, but will have a shiny, glossy appearance, and each 
fibre will be invested with a portion of dried coagulum or 
clot. 

The most simple plan of procedure^ when small parti- 
cles of coagulum can be removed, is to place them on a 
glass slide, and then breathe over them several times. A 
cover-glass is applied, and if the blood be present, a red 
margin will soon appear, and by the aid of a quarter-inch 



EXAMINATION OF BLOOD. 347 

lens the red blood corpuscles may be recognized. Should 
this be unsuccessful, a very small drop of water should be 
run in under the glass — any excess must be avoided, or the 
corpuscles will swell and lose their characteristic appear- 
ances. 

Such a simple method, however, is not always practi- 
cable. The stained portion of the material must then be 
cut out and macerated in a small quantity of equal parts 
of glycerine and water, or, better, in a mixture proposed by 
Hofmann — 

Water, 300 parts 

Glycerine, 100 " 

Chloride of sodium, 2 ** 

Corrosive sublimate, i part. 

If small portions of the fibres of the material be 
placed under the microscope, corpuscles will be seen either 
free in the fluid or entangled in the meshes of the fibres. 

A very important question often arises; granted that 
the stain is caused by blood, is it human ? At present our 
knowledge on this subject is very limited. In all animals 
with red blood the globules have a disc-like form. In the 
mammalia, with the exception of the camel tribe, the out- 
line of the corpuscle is circular. In this tribe, and in 
birds, fishes, and reptiles, they are oval in form. In the 
three last mentioned classes of animals they possess a cen- 
tral nucleus. We are able to say with safety, then, that 
blood corpuscles are either mammalian and may be human, 
or that, being oval in shape, the corpuscles come from one 
of the other classes above mentioned. 

Dr. Lionel Beale, in his work on ^^The Microscope in 
Medicine," figures a large number of corpuscles which are 
worthy of reference. 



CHAPTER XXII. 

CUTANEOUS PARASITES. 

In order to complete the account of the most important 
parasites found in disease, a short description must be 
given of those producing various affections of the skin. 

The clinical features are usually quite sufficient upon 
which to base a diagnosis, but the evidence afforded by the 
microscope will corroborate the opinion, and in a few 
atypical cases may yield the only means of arriving at a 
definite conclusion. 

Some of the bacteria producing cutaneous affections, 
such as those pathognomonic of tubercle, leprosy, etc., 
have already been considered. 

Cutaneous parasites may be divided into animal and 
vegetable. The latter will be considered first, as being the 
more important. 

Vegetable Parasites. 

Trichophyton tonsurans. — This is the character- 
istic fungus of '^ringworm." The diagnosis is not 
difficult clinically, the presence of short, broken-off hairs, 
in small patches, sufficiently indicating the nature of the 
complaint. 

As corroborative evidence one or two of the hairs should 
be removed with forceps and soaked for a little time in 
dilute liquor potassae. The hair seems to be covered near 
its roots with an asbestos-like covering of a dull v/hite 
color. After soaking, they are placed on glass slides with 

348 



CUTANEOUS PARASITES. 



349 



a little of the potash solution, a cover-glass applied, and 
light pressure made so as to separate the component fibres 
of the hairs. 

Attention should first be directed to the hair itself. 
The surface will be seen to be rough, the cortex and 

Fig. 78. 




Trichophyton tonsurans [Alder Si?iith.) 

medulla being almost indistinguishable. The free end, 
instead of being pointed, is ragged and split. The fun- 
gus (Fig. 78) consists almost entirely of spores ; these are 
circular in shape and about .004 mm. in diameter; they 
possess nuclei, and are chiefly arranged in chains, dis- 



350 MEDICAL MICROSCOPY. 

tributed between the disordered fragments of the hair, 
being particularly numerous about the root ; the my- 
celial threads are few, jointed, somewhat curved in 
their course, and small granules are seen in their interior. 
When attacking the head this disease is known as Tinea 
tonsurans ; when the body is affected it is termed Tinea 
circinata or Tinea marginata. 

Tinea marginata, formerly called ^^ eczema margina- 
tum," occurs only in adult males; the same fungus is 
found in all the forms. 

Microsporon furfur (Tinea versicolor). — The af- 
fection caused by this fungus is also known as Pityriasis 
versicolor. It occurs as yellowish-brown spots, scarcely 
rising above the level of the skin, and distributed gener- 
ally over the body, especially over the chest and back. 

To demonstrate the fungus some of the scales should be 
removed and examined in a drop of dilute potash. If an 
opportunity should occur of obtaining sections of skin thus 
affected, very satisfactory specimens may be obtained by 
staining them with Weigert's modification of the Gram 
method (see p. 102). The spores are found collected to- 
gether into heaps, and are a little larger than those of 
Tricophyton tonsurans. The mycelial threads are very 
numerous and form a thick, wavy mass, between the com- 
ponent parts of which the spores can be seen. 

Achorion Schonleinii. — If a small piece of the crust 
from a case of favus (Fig. 79) is examined in liquor po- 
tassae, dense masses of mycelia will be seen ; they are often 
so thick that the spores can scarcely be made out. These 
latter are oval or circular in shape and a little smaller than 
those of Tricophyton tonsurans. 



CUTANEOUS PARASriES. 



351 



Animal Parasites. 
Pediculi. — Three species of lice are parasitic on man : 
(i) Pediciilus capitis; (2) Pediculns vestimenti vel corporis; 
(3) Pediculus pubis. 




I. Pediculus capitis. — In all' cases of pustular inflam- 
mation of the scalp the hair should be carefully searched 
for pediculi, although the impetigo which results is pro- 



352 MEDICAL MICROSCOPY. 

duced more by the scratching of the patient than by the 
irritation of the lice. 

Children are most frequently attacked, and women more 
than men. 

The louse is about a line in length, of a dirty-white 
color, and covered with short, scattered hairs. The abdo- 
men is oval, and distinct from the head and thorax. The 
head has two short antennae, and large, prominent black 
eyes. The parasite has six well-developed legs, furnished 
with strong claws. The male, which is rather smaller than 
the female, has a peculiar long projection on its back — the 
penis. The animal itself cannot always be found, but 
equally diagnostic are the presence of ^^nits.'' These 
contain the ova, and are small, white, semi-transparent 
bodies, made of a hard material and triangular in form. 
They are firmly attached to the hairs, about three-quarters 
of an inch from the roots, by short pedicles, being glued to 
them by a material secreted from the lice. 

2. Pediculus vestimenti. — In general appearance 
this louse closely resembles the pediculus capitis ; it is, 
however, rather larger in size. Its ova are deposited in 
the clothing and not on the surface of the body. 

3. Pediculus pubis. — As its name indicates, this par- 
asite chiefly attacks the hair about the generative organs. 
It is different in shape to the other two varieties, resem- 
bling a small crab. It is also smaller, and the line of 
separation between the abdomen and thorax is not so well 
marked. The legs are short and curved, and terminated 
by strong claws. 

Sarcoptes hominis. — This parasite, also known as 
the Acarus scabiei, or itch mite, produces the well-known 
disease, scabies. The clinical features are too well known 
to need description here, and we need only consider the 
parasite itself. 



CUTANEOUS PARASITES. 353 

The male insects are but rarely seen, as they do not 
burrow, but live upon the surface of the body. The female, 
after impregnation, makes her way under the skin, forming 
the characteristic cuniculus or '' run." To demonstrate 
the mite, a burrow should be laid open with a stout needle, 
from the entrance to its blind extremity; the acarus will 
then be seen as a small white particle, and usually clings 
to the point of the needle. It should be transferred to a 
glass slide and covered with liquor potassae, or if it be 
desired to preserve it, the best medium for the purpose is 
glycerine jelly. 

The animal is in shape something like a tortoise ; it has 
four pairs of legs, and is covered with a chitinous integu- 
ment furnished with short spines and scattered hairs. The 
female is larger than the male, and on the four anterior 
legs suckers will be noticed. The male has suckers on all 
the legs. 

Another method for more completely examining the 
mite and its burrow is to excise the burrow, parasite and 
all, by means of a sharp pair of scissors, although this is 
rather a difficult little operation. By means of a good 
lens, the passage can then be seen, often containing a row 
of oval eggs in chitinous cells, with the female acarus lying 
at one end. 

Acarus foUiculorum. — This is a small parasite oc- 
casionally found in the hair follicles and sebaceous glands. 
It cannot be said to be pathological, as it is found in about 
ten per cent, of all healthy adults. 

The acarus may be demonstrated by squeezing out the 
contents of some of the follicles and placing them on a 
glass slide with dilute liquor potassse. The animal will 
then be seen to possess a pointed abdomen, distinct from 
the head and thorax, which are continuous. It has four 
pairs of legs, each terminating in three strong claws. 



CHAPTER XXIII. 
EXAMINATION OF FOOD AND WATER. 

The examination of drinking water is almost entirely 
conducted by chemical analysis, although an investigation 
of the sediment by the microscope, and especially by bac- 
teriological processes, may yield important information. 
The adulterations of food are detected partly by chemical 
and partly by microscopical methods. We shall naturally 
consider here only the points that can be determined by 
the use of the microscope ; these are so many that only the 
most important can be touched upon. If the student is 
desirous of going more deeply into this subject, he cannot 
do better than consult the work by Dr. A. H. Hassall, 
^^Food: its Adulterations, and the Methods for their De- 
tection." 

Examination of Food. 

In looking down the list of substances commonly used 
for the adulteration of food, the various cereals occupy a 
prominent place, and an intimate knowledge of their 
structure and the appearances of their grains is absolutely 
necessary for the purposes under consideration, and we 
shall therefore begin with a description of these bodies, 
afterward indicating the food-matters with which they may 
be found mixed. 

It may be here stated, however, that food is adulterated 
with three main objects — firstly, to increase its bulk and 
weight ; secondly, to improve its appearance and color ; 
thirdly, to add to its taste, smell, or other properties. 

354 



EXAMINATION OF FOOD AND WATER. 355 

Wheat. — Several structures enter into the formation of 
the grain of wheat, as well as that of the other cereals. 

The seed is surrounded by membranes, technically known 
as the ^* testa; " the surface of the seed consists of angular 
cells, filled with granular oily matter, whilst its substance 
is made up of cells filled with starch corpuscles, and it is 
with these latter that we are most closely concerned, as the 
testa is in great part removed in the process of grinding 
and dressing the flour, and the same may be said of the 
cells forming the surface of the grain. 

The general structure of a ^^ starch grain " is as follows : 




Wheat- starch Grains. 

Each granule exhibits a peculiar spot, termed the ^^ hilum," 
round which are seen a set of circular lines which are for 
the most part concentric with it. A characteristic effect is 
produced by polarized light : each grain shows a dark cross, 
the point of intersection being at the hilum. 

The starch grains of wheat (in fact, of all cereals) are 
best viewed with a quarter-inch lens. They will be seen 
to consist of round or oval particles of various sizes (Fig. 
80), some being very small and others of considerable size, 
whilst only a few are of intermediate dimensions. The 
small grains are chiefly circular in shape, with a central 



356 MEDICAL MICROSCOPY. 

hilum ; the larger granules form rounded or flattened discs, 
with thin edges ; in these the hilum and concentric lines 
are barely visible, if at all. 

Occasionally some of the larger granules are twisted or 
turned up at the edges, and when seen sideways present 
the appearance of a longitudinal furrow, giving a very good 
representation of a hilum ; but if the cover-glass be slightly 
moved, so as to cause the grains to turn over, their real 
nature is at once apparent. 

Portions of the outer envelopes of the wheat grain may 
be detected in the coarser and more branny flours. The 
testa consists of three layers of cells, two of which are 

P^TG. 8t. 

Barley- Starch Grains. 

disposed longitudinally and the third transversely to the 
axis of the seeds. 

The cells forming the surface of the seeds are large and 
angular. 

Barley. — Like that of wheat, barley-starch (Fig. 81) 
consists of small and large grains ; in fact, the two starches 
are almost indistinguishable from one another. The larger 
grains are, as a rule, more distinctly ringed, while a greater 
proportion of them present the longitudinal furrow. If 
portions of the testa can be secured, the difl*erence from 
wheat is seen to be more marked. The testa of the barley 
grain consists of four layers of cells, smaller than those of 
wheat. There are three layers of longitudinal cells which 
are not beaded as in the case of wheat. 



EXAMINATION OF FOOD AND WATER. 357 

The cells of the surface of the grain are not nearly so 
large as those of wheat, and there are three layers instead 
of one. 

Rye. — In general form the starch grains resemble those 
of wheat, but a few differences may be noticed. The lesser 
grains are decidedly smaller than the corresponding grains 
of wheat, and many of the large rye granules have a pecu- 
liar rayed hilum. With the polariscope they exhibit a 
very strongly-marked cross, thus distinguishing them from 
barley-starch. 

The testa of rye also closely resembles that of wheat. 

Oatmeal (Fig. 82). — The starch grains are smaller than 
those of wheat and more uniform in size ; they are polyg- 

FlG. 82. 

Oatmeal-Starch Grains. 

onal in shape and no hilum or concentric rings are visible. 
They tend to cohere together, forming rounded masses 
presenting a reticulated surface. With polarized light, 
unlike the starches of other cereals, no crosses are produced. 
The longitudinal cells of the testa are large and well- 
defined, and long and pointed hairs arise from some of 
them. The transverse cells form a single layer and are 
almost square in shape. 

Maize (Indian corn flour). — The starch grains (Fig. 
S^) are polygonal in outline^ and exhibit well-marked 
central depressions, and occasionally a radiated hilum. 
They are larger in size than those of the oat, and do not 




358 MEDICAL MICROSCOPY. 

tend to cohere into masses. With the polariscope a 
well-defined cross is visible. 

The testa is made up of two membranes, the outer 
consisting of seven or eight layers of cells which are 
much longer than they are broad, and the margins of 

Fig. 83. 

S O is/ 

Maize- starch Grains. 

the outermost layer are beaded. The inner membrane 
consists of a single layer of cells. 

The cells containing the starch granules are very 
angular and are subdivided by numerous septa. 

Rice (Fig. 84). — In shape the starch grains resemble 
those of the oat, being polygonal; they are, however, 

Fig. 84. 




<o r " 



a 



° <^ o 00 



Rice-starch Grains. 



much smaller. They exhibit well-marked central de- 
pressions and raised edges. 

The testa is rather complex. The outer surface is 
thrown up into ridges, arranged both longitudinally and 
transversely. The ridges contain silica in the form of 
granules. The substance of the husk is composed of short 
fibres, arranged like the ridges. Beneath this fibrous mem- 



EXAMINATION OF FOOD AND WATER. 



359 



brane is a thin layer of angular cells, which are longer 
than they are broad. 

Beans (Fig. 85).* — Bean-starch cells are more or less 
oval and somewhat flattened ; they exhibit a longitudinal 
cleft in the centre of the grain, crossed by transverse fis- 
sures. 

Peas (Fig. 86). — Pea-starch grains are smaller than 
those of the bean *and less flattened, but otherwise similar 
in shape. The longitudinal cleft which runs the whole 
length of the grain does not show the transverse fissures. 



Fig. 85. 



Fig. 86 




Bean -starch Cells. 




Pea-Starch Grains. 



Sago (Fig. 87). — The starch is obtained from the pith 
of several kinds of palms. The starch grains are elon- 
gated in form and of considerable size; they are larger at 
one end than at the other. The large end is rounded, and 
the other truncated. The hilum is usually circular, but 
sometimes cracked, when it appears as a slit or star. In 
some of the granules a few concentric rings may be seen. 

Tapioca (Fig. 88). — The starch is procured from the 
root of the plant. The granules are small in size, some- 
times united into groups of three or four. They are 

* Figures 80-94 are borrowed, with the kind permission of the author, 
from Parkes' '* Hygiene and Pubhc Health." 



360 



MEDICAL MICROSCOPY. 



roughly circular in form, although owing to mutual pres- 
sure they may become truncated at one extremity, as with 
the sago grains. The hilum is round and well marked. 

Arrowroot (Fig. 89). — There are several kindsof arrow- 
root. The starch grains of each variety differ slightly 
from each other, but it would be of no advantage to de- 
scribe each form separately. Generally speaking, the 
grains are oval or pyriform in shape ; "the corpuscles of 
^^ British arrowroot " are often circular. Their size varies 
greatly, but they are usually large. The hilum is situated 
in the broad extremity of each granule, and the concentric 




© 



Sago-starch Grains. 



Fig. 88. 






Tapioca-Starch Grains. 




lines are fine, regular, and, although crowded together, are 
well marked. 

Potato (Fig. 90). — The starch grains of potato are 
very characteristic. They are of large size. Most of 
them are pyriform in shape, others almost circular. The 
hilum is situated at the narrow end. The striae are well 
marked, but are not so regular or so numerous as in arrow- 
root starch, and the lines appear to be coarser. 

Turmeric. — This substance is easily recognized by its 
bright yellow color. When crushed, in addition to almost 



EXAMINATION OF FOOD AND WATER. 



361 



Structureless debris, starch granules, pyriform in shape, 
with well-defined hilum and concentric lines, are seen. 

This completes a description of the various substances 
used in the adulteration of the chief articles of food, and 
we have now to indicate the principal articles of diet 
which are adulterated by them. 

Arrowroot. — The best kind of arrowroot is generally 
considered to be the Maranta or West Indian. The starch 
grains are usually oval in form, but sometimes almost tri- 
angular ; they vary greatly in size. The hilum is situated 
at the broad end of the grain, whilst in the inferior kinds 
of arrowroot it is found at the narrow end. It is not 



Fig. 89. 





Arrowroot- starch Grains. 



Potato-starch Grains. 



always circular, but is frequently seen as a short, sharp line 
running transversely. In the other kinds of arrowroot the 
hilum is nearly always circular, so that by the forhi and 
position of the hilum Maranta arrowroot can be readily 
distinguished from the starches used to adulterate it. 

In order to increase its bulk and weight, arrowroot is 
mixed with ^^ sago, potato, and tapioca starches, and vari- 
ous mixtures and combinations of these with the inferior 
arrowroots" (Hassall). With a little practice, all these 
substances can be recognized with the microscope. 

Bread. — Bread should be made of pure wheat flour, but 
31 



362 ' MEDICAL MICROSCOPY. 

many inferior forms are often added in order to increase its 
bulk and weight. The most common adulterations are 
mashed potatoes, rye, maize, rice, and beans. Alum and 
salt are also often added in large quantities, but they can 
only be detected by chemical analysis. 

Flour. — Wheat flour is chiefly adulterated with rice, 
beans, maize, rye, and potato-flour. The microscope 
forms the only means of recognizing these impurities. 
Alum and other inorganic salts are sometimes added in 
large quantities. 

Wheat and other cereals are liable to become infected, 
and consequently deteriorated, by the development of 
various parasites, animal and vegetable. 

Smut {Uredo segetic??i). — This disease forms yellow spots 
upon the stem, leaf, and chaff. When a scraping is made, 
placed under the microscope, and examined under a quar- 
ter-inch lens, minute sporules are seen, of granular aspect, 
and many of them presenting a double contour. 

Bunt {Uredo foetidd). — The spores are larger than those 
of ^' smut," and exhibit a reticulated appearance. This 
fungus only attacks the grains of wheat. 

Rust {Puccinia graniinis'). — The spores form yellower 
brownish oval spots upon the stem and leaf. The spores 
are intermediate in size, between those of ^* rust " and 
*^smut." In the earliest stage of growth they are round, 
but afterward become oval, and attached by a short, slen- 
der stalk to the surface on which they develop ; after a 
time they become free. 

Ergot (^Oidium arbortifaci ens), — When ergot afl'ects the 
grain, the seed coat and starch cells are replaced by a layer 
of dark cells, the large cells by the small cells of the ergot, 
and the starch grains by drops of oil. A bloom appears 
on the grain, and consists of the sporidia of the fungus. 
If wheat be badly stored or allowed to become damp. 



EXAMINATION OF FOOD AND WATER. 363 

various moulds and fungi form, such as imicor mucedo^ 
penicillium glaucinn, and aspergillus niger. 

Ear-cockle (^Vibrio tritici), — This parasite fills the grain, 
which becomes green and then black, with a cotton-like 
substance. Under the microscope this material is seen to 
be composed of numerous slender animalculae, which, when 
placed in water, exhibit the most active movements. 

In addition are frequently found Acarus farince. (Fig. 
91) and the weevil, or Calandra granaria, a small insect, 
visible to the naked eye, which eats the core out of the 
grain, leaving only the shell. 

OatmeaL — The characters of the starch grains have been 




Acarus farinae. 

described on page 357. ^^It would hardly be supposed 
that sufficient inducement exists for the sophistication of an 
article like oatmeal ; it appears, however, that the supposi- 
tion is not correct. ... Of thirty samples of oatmeal 
submitted to examination, sixteen, or rather more than one- 
half, were found to be adulterated with large quantities of 
barley meal" (Hassall). Barley costs about half as much 
as oats, hence the inducement. 

The investing membranes of the oat, barley, and wheat, 
technically termed ^' rubble " or '^ sharps," are also added. 

The testa of the various cereals have been described in 
the early part of the chapter, and it is mainly on their 



364 MEDICAL MICROSCOPY. 

recognition by the microscope that the detection of the 
adulteration depends. Rice and maize are sometimes em- 
ployed in addition to the cereals named above. 

Beverages. 

Tea. — The structure of the tea leaf is characteristic, 
although it varies somewhat according to age. It is oval 
in shape and has a serrated border; the primary veins run 
out alternately from the mid-rib, and turn toward the 
point of the leaf, but without reaching the border. 

The chief adulteration formerly consisted in mixing 
other leaves with those of the tea plant, but since the duty 
has been greatly reduced and the Customs authorities have 
their own appointed analysts, such adulteration is now 
almost unknown. Mineral matters are sometimes largely 
employed, such as sand, quartz, and magnetic oxide of 
iron. 

For the detection of these adulterations chemical means 
are usually employed, but it is also necessary to have a 
thorough knowledge of the structure of the tea leaf and 
of the chief leaves that may be mixed with it. Extensive 
practice alone can give the necessary information. 

Coffee. — We are concerned only with the structure of 
the coffee berry. The testa, or investing membrane, is 
chiefly made up of elongated oval cells (Fig. 92), adherent 
to one another, and presenting irregular cross markings on 
their surfaces ; they form a single layer and rest upon a 
thin membrane having an indistinct fibrous structure. 

The substance of the berry consists of an irregular net- 
work of fibres forming a cellular structure, in which is con- 
tained a considerable quantity of oily matter. 

The most prevalent adulterant of coffee is chicory. 

Chicory is the root of a plant, not the seed. It can be 
fairly readily distinguished from coffee under the micro- 



EXAMINATION OF FOOD AND WATER. 



365 



scope. The ducts have a strong resemblance to the cells 
of the testa of coffee, but they present a dotted surface 
(Fig. 93) which sufficiently distinguishes them. The chief 



Fig. 92. 



Coffee ; Cells of Testa and Cellular Structure. 




part of the root is made up of cells embedded in a coarse 
areolar tissue. The accompanying figures show the main 
differences between the two structures. 



Fig. 93. 




Chicory. Dotted Ducts and Cellular Structures. 

Coffee, in addition, is adulterated with roasted wheat, 
rye, and potato flour, roasted beans, mangel wurzel, and 
acorns, and to improve its color burnt sugar is sometimes 
employed. 



^66 MEDICAL MICROSCOPY. 

Cocoa. — The starch grahis of cocoa are very small and 
circular in form (Fig. 94). When the seed is compressed 
it breaks up into pieces, which are known as ^^nibs/' 
and within the cells forming these the starch grains are 
found. 

The chief adulterants of cocoa are various forms of 
starches, namely, Maranta, East India, and Tahiti arrow- 
root ; the flours of wheat, maize, sago, potato, and tapioca; 
sugar, chicory, and cocoa husks. All these substances may 
be recognized by the microscope. 

To improve its color, Venetian red, red ochre, and other 
ferruginous earths are added. 



Fig. 94. 



0^ 
C7 0" Q 



O o 



Q 




3 



Cocoa-starch Cells. 

Examination of Drinking Water. 

As already stated, chemical analysis is by far the most 
important means of examining drinking water, but not 
infrequently the nature of the suspended matters have 
to be determined, and this is accomplished with the micro- 
scope. 

When the water is turbid, no difficulty will be found 
in obtaining a sediment, but, as a rule, the deposit is 
slight. 

Dr. J. D. MacDonald ('^ A Guide to the Microscopical 
Examination of Drinking Water") gives the following 
directions for the collection of specimens : ^^ A tall glass 



EXAMINATION OF FOOD AND WATER. 367 

vessel is taken, and filled up to the neck with the water to 
be tested. A circular disc of glass, resting upon a hori- 
zontal loop at the end of a long wire, is let down to the 
bottom of the vessel, which is then covered and set aside 
for twenty-four or forty-eight hours. At the end of this 
period the water is siphoned off with a piece of India-rubber 
tubing, leaving only a thin stratum over the glass disc. 
This is now carefully raised and laid upon a piece of blot- 
ting-paper, so as to dry its under surface, when it may at 
once be transferred to the stage of the microscope, with a 
large piece of thin covering glass so placed as to exclude 
all air-bubbles.'' 

Another good plan is not to siphon off all the water, but 
to leave a little, then shake the deposit up with it, and 
pour the mixture into a tall conical glass, from which por- 
tions of the sediment may be taken up by means of a 
pipette and examined in the usual way. 

Various mineral substances and dead vegetable and ani- 
mal matter will then be observed, together with many living 
forms, both animal and vegetable. 

We cannot enter here into a description of the numer- 
ous objects which present themselves in various samples 
of water, for to do so would entail several additional chap- 
ters. The reader who is interested in such matters cannot 
do better than consult Dr. J. D. Macdonald's book, already 
alluded to, where he will find a full description of a large 
number of organisms and a series of beautifully executed 
lithographic plates. 

Bacteriological Methods are also now largely em- 
ployed in the examination of drinking water. The sam- 
ples are collected in sterilized flasks plugged with cotton- 
wool. 

The apparatus for plate cultivations (see p. 384) must be 
prepared, shallow cultivation dishes being very convenient. 



368 MEDICAL MICROSCOPY. 

A tube of liquefied nutrient gelatine is taken, and the 
plugs of both it and the flask having been removed, a drop 
of water is transferred to the tube by means of a sterilized 
graduated pipette, the plugs being immediately replaced. 
The tube is rotated so as to diffuse the organisms through 
the gelatine, and a plate cultivation is then made. When 
the gelatine has set, the plate is transferred to a damp cham- 
ber. Colonies will commence to develop in two or three 
days and may then be examined. 

^^ Drop cultivations " may also be made in sterile bouil- 
lon. 

With plate cultivations the number of colonies which 
develop are usually counted, so as to form some estimate 
of the number of micro-organisms in a measured quantity 
of the water under examination. For this purpose a glass 
plate, ruled by horizontal and vertical lines into centimetre 
squares, some of which are again divided into ninths, is so 
arranged on a wooden frame that it can cover the plate 
without touching it. The colonies in a certain number of 
squares are counted, so that the number of colonies on 
the entire surface can be calculated by a process of multi- 
plication. 

Cover-glass preparations should be made from the col- 
onies and stained in the usual way. 



CHAPTER XXIV. 

BACTERIOLOGICAL METHODS. 

Before a micro-organism can be considered to be patho- 
genic it has to satisfy certain conditions. These have been 
laid down by Koch as follows : — 

1. The micro-organism must be found in the blood or 
other fluids or tissues of the animal suffering from the dis- 
ease in question, and in no other disease. 

2. The micro-organisms must be isolated and cultivated 
on nutrient media outside the body. 

3. A pure cultivation thus obtained must produce the 
same disease when introduced into the body of a healthy 
animal. 

4. The same micro-organisms must be found in the ani- 
mal inoculated. 

In order to carry out the investigations necessary for 
demonstrating the above postulates certain apparatus is 
required. Bacteriology is essentially an exact science, and 
unless every single detail is carefully adhered to in the 
various processes adopted, failure can be the only result. 
The apparatus that is absolutely needed is more compli- 
cated than that required for the ordinary pathological 
methods which have been described in the previous chap- 
ters. A good gas and water supply must be at hand, and 
for any really satisfactory work a room should be set apart 
for the purpose ; in other words, a well-appointed laboratory 
is almost indispensable. 

It is not proposed in this chapter to enter deeply into 
32 369 



370 MEDICAL MICROSCOPY. 

the methods of bacteriological research — for this purpose a 
large volume would be needed — but only such procedures 
and apparatus will be described by which micro-organisms 
can be isolated and cultivated in the simplest way. 

It is difficult to detail on paper the delicate manipula- 
tions which have to be learnt, and I should strongly advise 
the student who wishes to study the science of bacteriol- 
ogy, wherein lies a huge field for investigation, to com- 
mence by taking out a ^^ course " of lessons at one of the 
laboratories now open for the purpose. 

In order to carry out original investigation a sound 
knowledge of the rudiments of the science is necessary, 
and this can only be obtained by practical study and 
experience. 

Apparatus Required. 

Steam sterilizer. 

Hot-air sterilizer. 

Hot-water filter. 

Incubator. 

Test-tubes. 

Glass flasks. 

Large cultivation dishes. 

Shallow cultivation dishes. 

Platinum needles. Scalpels. 

Funnels; filter-paper, etc. 

Steam Sterilizer (Fig. 95).^ — This consists of a 
cylindrical vessel made of leaded iron with a copper bot- 
tom. It is about two feet in height, and is jacketed with 
thick felt. The lid is conical, has handles on either side, 
and is perforated at the apex to hold a thermometer. On 

* Figures 95-101 are borrowed, with the kind permission of tlie 
author, from Professor Crookshank's '' Manual of Bacteriology." 



BACTERIOLOGICAL METHODS. 



371 



one side is a gauge which marks the level of the water, 
and a tap for running it off. Inside the vessel is an iron 
grating about two-thirds of the way down which separates 
the water from the steam chamber. The apparatus stands 
upon three legs, and is heated from below with a Fletcher's 
burner or two or three Bunsen's. When the water com- 

FiG. 95. 




Koch's Steam Sterilizer. 



mences to boil, the lid is removed and the flasks or tubes 
with their contents, which have to be sterilized, are placed 
within it, resting on the grating. Test-tubes are usually 
arranged in a wire basket, so that a large number may be 
stacked together. 

Hot-air Sterilizer. — This is a square box, made of 
sheet iron, with double walls ; it may either be hung 



372 



MEDICAL MICROSCOPY. 



against the wall or be supported on four legs. In the roof 
is a hole for the thermometer, and it is heated from below 
by a Fletcher's burner. The temperature can be raised to 
150° C. Cotton-wool, test-tubes, flasks, etc., may be ster- 
ilized in it, being kept at a high temperature for about an 

Fig. 96. 




Hot-water Filtering Apparatus with Ring Burner. 



hour. Cotton-wool is most conveniently enclosed in glass 
capsules. 

Hot-water Filter (Fig. 96). — This apparatus con- 
sists of a copper funnel with double walls, the space be- 
tween them being filled with water, which is kept hot by 



BACTERIOLOGICAL METHODS. 373 

means of a circular burner, which at the same time serves 
as a funnel ring. 

Within the copper cone fits a glass funnel, which is fixed 
by means of a caoutchouc plug. It is employed for filtering 
the solid cultivating media, which are liquid when hot. 

Another arrangement is to have a metal receiver con- 
taining the funnel placed in connection with the steam 
sterilizer, which thus serves a double purpose. 

Incubator. — Most incubators are rectangular chests 
with double walls, the interspace being filled with water 
and kept at a constant temperature by means of a *^gas 
regulator." There are several varieties, but the one known 
as *^ Babes'" is about the best (Fig. 97). The sides and 
roof are covered with felt. In the roof are two hooks, 
one for a thermometer and the other for the gas regulator. 
The front of the chest is provided with a glass door and a 
sliding glass panel, which are likewise closed in by a sheet 
of felt. It is heated from below by two protected burners, 
which are supplied with gas through a '^ thermo-regulator." 

The best form of regulator is Reichert's, a full descrip- 
tion of which will be found in the text-books on Bacteri- 
ology. 

Test-tubes. — A large number will be required. The 
most convenient size is about six inches long by five-eighths 
of an inch in diameter. 

Glass Flasks. — Large flasks are employed for boiling 
nutrient media, and small conical ones for storing solid 
cultivating material. 

Large Cultivating Dishes. — These are chiefly used 
for potato cultivations, also for ^* plate cultivations." They 
consist of two parts, the larger and upper fitting over the 
smaller and lower. 

Small Cultivating Dishes. — These are much smaller 
and shallower than the preceding • they should be procured 



374 MEDICAL MICROSCOPY. 

as flat as possible, as they are employed instead of plates 
for isolating colonies of bacteria. 

Platinum Needles (Fig. 98). — These are easily made 
with stout platinum wire and glass rods. The wires — 
about two or three inches in length — are forced into the 
ends of the rods by heating the ends of the latter until soft 

Fig. 97. 




Babes' Incubator. 

enough to admit them. At least three will be required : 
one having a loop at its extremity, another being bent 
into a short hook, whilst the other is allowed to remain 
straight. 

Potato Knife. — This consists of a broad, smooth- 
bladed knife set in a wooden handle. 



BACTERIOLOGICAL METHODS. 



375 



The other accessory apparatus need not be described, 
and may be procured as required. 

In all matters connected with the microscope, cleanliness 
cannot be too strongly insisted upon, but with bacteriologi- 
cal work it is, if possible, even more important, for with- 
out it, it is impossible to proceed. All apparatus and 
instruments must not only be clean to the naked eye, but 
must be thoroughly purified from all germs. 

This is accomplished mainly by heat. The word that 
must be constantly dinned into the ear of the beginner is 
^^ sterilized Metal instruments may simply be heated to 

Fig. 98. 




Platinum Needles ; Straight, Hooked, Looped. 



redness in a Bunsen burner. The scalpels must afterward 
be placed with their handles on the table and their blades 
projecting over the edge. It should become a routine 
practice ahvays to heat the platinum wares both before and 
after use. 

Cotton-wool should be sterilized by being placed in the 
hot-air sterilizer. 

Glass apparatus, test-tubes, flasks, etc., after being thor- 
oughly washed, should be swilled out with distilled water, 
next with corrosive sublimate solution (i in 1000), then 
rinsed with alcohol and ether, after which they are plugged 



376 MEDICAL MICROSCOPY. 

with cotton-wool and placed in the hot-air sterilizer for an 
hour, when they may be considered ready for use. 

Cultivating Media. 
A very large number of cultivating media have been 
introduced, but only those most commonly used wnll be 
described here. They are of two kinds, solid and liquid. 

Solid Media. 

Potato Cultivations. — This is the most simple form 
of cultivating medium. 

The usual way of conducting the process is as follows : — 

Smooth-skinned potatoes should be chosen, as free as 
possible from ^^eyes" and rotten spots; should any of 
these be present they must be picked out with the point 
of a knife. Each potato is scrubbed with a hard brush 
and then soaked for half an hour in a five per cent, solu- 
tion of corrosive sublimate. 

They are next placed in a ^^ potato receiver" and 
heated in the steam sterilizer at a temperature of 100° 
for about half an hour, after which they are allowed to 
cool. 

Meanwhile, the ^^ damp chambers" or large cultiva- 
tion dishes are prepared in the following manner : The 
glass is cleaned and washed in corrosive sublimate solu- 
tion as above described. A sheet of filter paper is cut 
to the size of the receiver, and after being dipped in the 
corrosive sublimate is placed in the bottom of the dish, 
the cover being at once replaced. The potato knife and 
two or three scalpels are also sterilized. 

The operator should then wash his hands and swill them 
with sublimate solution. 

When the potatoes are cool, an assistant raises the top 
of the potato receiver, and a potato is picked out and held 



BACTERIOLOGICAL METHODS. 377 

between the fingers and thumb of the left hand. By a 
clean cut with the large knife, the potato is almost divided 
into two. The assistant now raises the upper part of the 
cultivating dish, and the potato is quickly placed on the 
damp paper, a final turn of the knife separating the two 
parts, so that they lie side by side, cut surface uppermost ; 
the dish must be immediately covered. A second potato 
is then treated in the same way, all four portions being 
left in the same receiver. 

After again cleansing the hands, one portion of potato 
is removed from the dish, and a small portion of the 
substance to be inoculated placed on the centre with a 
sterilized platinum needle. With one of the prepared 
scalpels, the material is distributed over the surface of 
the potato, a clear margin of about a quarter of an inch 
being left. The potato is replaced and another half 
taken up, the receiver being closed in the intervals. 
With another scalpel a small portion is removed from 
the inoculated surface of the first potato and spread over 
the surface of the second, and the process is repeated 
from the second to the third, and from the third to the 
fourth, so procuring ^^ first," '^ second," and third" 
attenuations. The inoculations are then left to grow. 

Another method of potato cultivation which is now 
much employed is to cut the potato after cooking into 
small squares (of course, with a sterilized knife) and 
place them in large test-tubes, and inoculate them with 
a platinum wire, or to cut small cubes and keep them in 
small covered capsules, which have been cleansed and 
sterilized. 

Nutrient Gelatine. — Haifa kilogramme (one pound) 
of fresh beef is taken, as free from fat as possible, cut 
into small pieces, passed through a mincing machine, 
and placed in a large flask. A litre of distilled water 



378 MEDICAL MICROSCOPY. 

is added, the whole well shaken and placed in a re- 
frigerator or ice-pail for twenty-four hours. It is then 
strained through linen, and the red juice thus obtained 
placed in a measuring glass and made up to a litre by 
the addition of more distilled water. To the resulting 
liquid, contained in a large flask, are added : lo 
grammes of dried peptone, 5 grammes of common salt, 
and 100 grammes of best gelatine. The mixture is gently 
heated in a water bath, and in a short time the gelatine 
will be dissolved. The flask should be occasionally shaken. 

When this part of the process is complete, the fluid must 
be neutralized or rendered slightly alkaline. For this 
purpose several pieces of red litmus paper should be 
laid on the table, and a concentrated solution of car- 
bonate of soda added to the mixture, drop by drop, a 
glass rod being frequently dipped into the flask, and then 
one of the pieces of litmus touched with it until the red 
paper becomes faintly blue. If too much alkali has been 
added, the error may be rectified with a little lactic acid. 

The liquid is next heated in a water bath for an hour, 
its reaction being tested once or twice during that 
period. Afterward it is filtered through paper, the hot 
water filter being used for the purpose. A pale straw- 
colored filtrate should result ; unless perfectly clear, it 
must again be passed through the filter. 

Whilst still hot it is poured into test-tubes and flasks 
(which have been cleaned and sterilized in the manner 
already described). A sterilized funnel should be em- 
ployed for the test-tubes, so as to prevent the mixture 
touching the upper part of the tubes, which should be 
filled for about a third of their depth, and the cotton-wool 
plugs immediately replaced. 

When filled, the tubes are placed in their basket, 
and, with the flasks, heated in the steam sterilizer for 



BACTERIOLOGICAL METHODS. 379 

ten minutes, this being repeated on two successive 
days. On the third day, whilst still hot, some of the 
tubes should be inclined, so that the medium will solidify 
diagonally along part of the tubes, instead of vertically. 

The tubes and flasks are now ready for inoculation. 

Nutrient Agar-agar. — This preparation remains solid 
up to a temperature of about 45° C. The first part of its 
preparation is conducted in the same way as was described 
in the preparation of nutrient gelatine, that is to say, the 
meat is minced, allowed to stand in distilled water for 
twenty-four hours, and then filtered. Instead of adding 
gelatine, however, twenty grammes of agar-agar are 
added to the litre of meat infusion. The solid sub- 
stance should be cut up into small pieces with scissors 
and then dropped into the flask. After boiling for an 
hour, the mixture is filtered through flannel, the hot 
water apparatus being again employed. It will probably 
be necessary to filter twice, and even then it is difficult 
to obtain a perfectly clear fluid ; but if only a slight 
cloudiness appears, it is fit for use. A small quantity 
of liquid collects in the tubes after a time ; this is 
no disadvantage, but care must be taken not to allow 
it to touch the cotton-wool plugs. Both ^^ straight" and 
'* oblique" tubes should be prepared. The tubes must 
be sterilized by being placed in the steam sterilizer for 
forty-five minutes on three successive days. 

Glycerine Agar-agar. — This medium is particularly 
used for cultivating tubercle bacilli. It is prepared in 
the same way as agar-agar, with the exception that after 
the mixture has been boiled, five per cent, of glycerine 
is added, and the whole is then filtered. 

Sterilized Blood Serum. — Koch's method of pre- 
paring this medium is as follows : Two or three glass 
cylinders are cleansed and sterilized in the way already 



380 MEDICAL MICROSCOPY. 

described. They should be fitted with glass stoppers, 
which ought to be greased with vaseline. The part of 
the animal's skin from which the blood is to be obtained 
is shaved, and thoroughly cleansed by washing with cor- 
rosive sublimate solution and a mixture of alcohol and 
ether. With a sterilized scalpel a blood-vessel is opened, 
and after the first jet of blood has escaped, the remainder 
is allowed to flow directly into the cylinders, which when 
full, and the stoppers replaced, are deposited in a refriger- 
ator or ice-pail for twenty-four hours. By that time the 
clots will have separated, and the clear amber-colored 
serum is drawn off in sterilized pipettes and placed in 
sterilized test-tubes, which are plugged with cotton-wool. 
The tubes should be filled about a third of their length, 
and they are then put into a sterilizer, at a tempera- 
ture of 58°, for about two hours, and this process re- 
peated on three successive days. The serum has now 
to be solidified, and for this purpose the tubes are 
arranged on a specially constructed apparatus, consist- 
ing of a tin vessel with double walls, between which hot 
water is kept, and covered on the top with a plate of 
glass. When laid on it the tubes are inclined so that 
the fluid solidifies obliquely. The temperature is raised 
to between 65° and 68"^ C, and the tubes carefully 
watched. Directly solidification takes place they must be 
removed, and the medium should appear transparent and 
of a pale straw color. 

Liquid Media. 
The liquid cultivating media are not so much employed 
for bacteriological research as the solid media. They have 
serious disadvantages ; in the first place, many pathogenic 
organisms will not grow well in them, and secondly, it is 
difficult to be sure that the cultivation is a pure one. 



BACTERIOLOGICAL METHODS. 381 

Much may, however, be learned from their use, especially 
in '* drop-cultures " and in some inoculation experiments 
on animals. Such a medium is also largely employed 
when it is desired to undertake a chemical examination of 
the culture, and by passing it through a porcelain filter to 
separate the bacteria from the cultivating fluid, and so ob- 
tain only their products. 

Bouillon. — Beef, pork, or chicken may be the basis 
from which the bouillon is made. The meat is cut up and 
minced in the same way as for nutrient gelatine, mixed 
with distilled water in the same proportions, the infusion 
allowed to stand in ice for twenty-four hours, filtered 
through linen, and the filtrate brought up to the litre with 
more distilled water; lo grammes of dried peptone and 
5 of common salt are then added. The mixture is neu- 
tralized with carbonate of soda in the way already de- 
scribed, and the flask of broth deposited in the steam 
sterilizer at a temperature of 100° C. for half an hour. 
The liquid is afterward filtered and the clear filtrate trans- 
ferred to sterilized test-tubes, which are plugged, and the 
medium sterilized by being placed in the steam sterilizer 
for half an hour on three successive days. 

Liquid Blood Serum. — This is prepared in the way 
already described, except that the final process for coagula- 
tion of the serum is omitted. A few artificial media are 
occasionally employed, of which the two following are the 
most important : — 

Pasteur's Fluid. — This liquid has the following com- 
position : — 

Pure cane sugar, 10 parts. 

Ash of yeast, .075 part. 

Ammonium tartrate, i part. 

Distilled water, 100 parts. 



382 MEDICAL MICROSCOPY. 

Cohn's Fluid. — This has been modified by Mayer and 
consists of: — 

Phosphate of potassium, .1 part. 

Sulphate of magnesium, i " 

Ammonium tartrate, 2 " 

Tribasic calcium phosphate, . 01 " 

Distilled water, 20 parts. 

Mode of Using the Solid Nutrient Media. 

Test-tube Cultivations. — The tubes should be kept 
in the dark until wanted, and then placed in a beaker, in 
the bottom of which a small quantity of cotton-wool has 
been placed. 

The cultivations are of two kinds, according to whether 
the gelatine has solidified obliquely or vertically. The 
former tubes are employed for surface cultivations, whilst 
the latter are used for *' deep growths." 

The mode of making the '' deep growths " will be de- 
scribed first. 

The cotton-wool plug is twisted out, so as to loosen any 
adhesions that may accidentally have formed, and held be- 
tween the fourth and fifth fingers of the left hand. The 
tube is held mouth downward in the same hand (Fig. 99). 
A straight platinum needle which has recently been ster- 
ilized and allowed to cool is dipped into the fluid contain- 
ing the bacteria, or a colony is removed from a plate culti- 
vation and the wire introduced carefully into the tube 
without touching the sides, and plunged once well into the 
jelly, quickly withdrawn, and the tube immediately 
plugged. Half a dozen tubes should be thus inoculated, 
and then put away in the dark and examined daily to ob- 
serve the growth of the cultivation. 

Surface Growths. — These are made in two ways : In the 
first, the plug is removed as before, and the sterilized 



BACTERIOLOGICAL METHODS. 



383 



Straight platinum wire is drawn once along the surface, 
after having been dipped into the fluid to be tested, or hav- 
ing on it a colony from a plate, or the results of a potato 
cultivation, etc., and the plug rapidly replaced. 

In the second method the substance to be inoculated is 
diluted so that the formation of colonies may be observed. 

About a drachm of normal saline solution is boiled in a 
sterilized test-tube, which is immediately plugged with 

Fig. 99. 




Method of Inoculating a Test-tube Containing Sterile Nutrient Jelly. 



steriHzed cotton-wool and allowed to cool. A small parti- 
cle of the pure culture or whatever material is under ex- 
periment is then introduced on a platinum wire and mixed 
with the solution by rotating the tube. A gelatine tube is 
now taken and the two held side by side as in Fig. 100. 
The two plugs are removed and held, one between the 
fourth and fifth and the other between the third and 



384 MEDICAL MICROSCOPY. 

fourth fingers of the left hand. A sterilized platinum wire 
is then dipped into the saline solution and drawn once 
across the surface of the gelatine, and afterward the plug is 
replaced. Several gelatine tubes should be inoculated from 
the same saline solution. 

A few additional details may be here added. It is 
rather inconvenient to hold the cotton-wool plugs as above 
directed, and the following procedure may be adopted: — 

A piece of gauze wire is heated to redness and placed 
on a tripod stand. The plugs are removed with sterilized 
forceps instead of with the fingers, and whilst the tubes are 
being inoculated are laid on the gauze wire. When they 
are again removed they are placed in the flame of the 
Bunsen-burner so that their external surfaces are scorched, 
the flame being extinguished by blowing it out, or better 
by the mere fact of their being placed back in the tubes. 

The plugs should be pushed down the tube until only 
a small piece projects ; this should be again set fire to, the 
flame extinguished by blowing and the mouths of the tubes 
covered with caoutchouc caps made for the purpose. 

The tubes should be labeled with the nature of their 
contents and the date of inoculation. If the culture has 
been taken from another tube, '^ second generation," or 
'^ first sub-culture," etc., should be added. 

Surface inoculations on agar-agar tubes and on blood 
serum are made in the same manner. 

Plate Cultivations. — Test-tube cultivations are best 
adapted for the growth of bacteria in pure cultures. In 
order to procure such cultures, '^ plate cultivations " have 
to be made in the following manner : The glass plates are 
similar to those used by photographers. They must be 
thoroughly cleansed, swilled in corrosive sublimate solu- 
tion, then in alcohol, and finally placed in iron boxes and 
heated in the hot-air sterilizer for several hours. Some 



BACTERIOLOGICAL METHODS. 



38s 



tubes of nutrient gelatine are placed in a beaker contain- 
ing warm water, so as to liquefy the gelatine, and the cot- 
ton-wool plugs loosened so that they can be taken out 
without delay. 

One of the tubes is now inoculated with some of the 
fungoid material by means of a sterilized platinum wire ; 
the needle should be rotated a few times so as to mix the 
bacteria thoroughly with the gelatine. Another tube is 
nov/ taken and the cotton-wool removed, the two tubes 

Fig. 100. 




Method of Inoculating Test-tubes, in the Preparation of Plate Cultiva- 
tions. 



being held side by side (Fig. 100). The plugs may either 
be laid between the fingers or laid on sterilized wire-gauze, 
as already described. A sterilized platinum wire, termin- 
ating in a loop, is now passed carefully into the first tube, 
and a drop removed and mixed with the gelatine in the 
second tube. The original tube is now plugged, and a 
third one opened and a drop removed from the second 
and placed in it, the operation being repeated with a 
33 



386 MEDICAL MICROSCOPY. 

fourth tube. The tubes must now be labeled thus : First 
tube, original ; second tube, ist dilution ; third tube, 2d 
dilution ; fourth tube, 3d dilution. The cotton-wool 
plugs should be temporarily replaced, with the necessary 
precautions, and the tubes placed in a beaker. 

The leveling apparatus is now made ready. This con- 
sists of a tripod stand with screw legs, so that by placing 
a spirit level on it, it can be made perfectly level. On the 
stand is placed a large glass dish containing iced water, 
and covered with a sheet of glass which has been sterilized. 
The glass apparatus may be replaced by a sheet of iron, 
which is rather to be preferred ; in the winter cooling may 
be dispensed with, but in the summer it must be laid on 
ice immediately before use. Damp chambers must also be 
prepared as described on p. 376. 

One of the glass plates (which have in the meantime 
been allowed to cool) is now removed from the box with 
sterilized forceps and laid on the glass or iron plate, and 
then covered with a bell-jar. 

The ^^ original'' tube is now taken and a part of the 
rim heated in the Bunsen burner. When nearly cool, the 
plug is removed and laid aside. With the left hand the 
bell-jar is raised and the contents of the tube poured on 
to the plate, the gelatine being distributed as evenly as 
possible within a short distance of the edge of the plate, 
by means of the part of the lip of the test-tube which has 
been heated. The bell-jar is replaced until the gelatine 
has solidified, when the plate is removed to the '' damp 
chamber" (Fig. loi) and laid on a ^^ bridge; " this con- 
sists of a glass plate about six inches long and two wide, 
supported at each end by two glass bars about half an inch 
thick. A similar bridge is laid over the plate resting on 
the lower support. 

Plates are prepared in a similar manner from the other 



BACTERIOLOGICAL METHODS. 



387 



tubes, a careful memorandum being kept of the order in 
which they were placed, or labels may be attached to the 
bridges. 

The cover must, of course, be replaced as each of the 
plates is deposited. When the series is complete the glass 
dish is set aside. In a short period, varying according to 
the nature of the bacteria, small colonies will commence 
to grow, gradually increasing in size and assuming their 
characteristic appearance. 

If the original culture was pure, only one form of col- 
ony will be seen, but if not, several varieties may be seen. 
On the '' original" plate they will probably be so numer- 
ous as not to be separable, but in the various dilutions they 

Fig. ioi. 




Damp Chamber for Plate Cultivations. 



will become more and more discrete, so that in the third, 
and perhaps also in the second, the individual colonies will 
be clearly separated from one another. 

One of the plates is placed on the stage of the micro- 
scope and examined with a low power. The colonies will 
appear more sharply defined if a small diaphragm is used. 
A characteristic colony is selected and arranged so that it 
occupies the centre of the field. 

A sterilized platinum wire, bent at the extremity into a 
small hook, is introduced between the object glass and 



388 MEDICAL MICROSCOPY. 

the plate. When the hook is seen close to the selected 
colony, the needle is dipped into it, raised, and withdrawn, 
carrying the whole or part of the colony with it, and a 
gelatine or agar-agar tube is immediately inoculated with 
It, or a cover-glass preparation may be made of it for the 
purpose of ascertaining the nature of the microbe. The 
above process is a little difficult at first, but wath some 
practice it can be accomplished with rapidity and precision 
and a pure culture assured. There are two modifications 
of *^ plate cultivations." 

Shallow cultivation dishes, carefully cleaned and steril- 
ized, may be used instead of plates, so avoiding the use of 
the leveling apparatus. One dish must, of course, corre- 
spond to each test-tube, and the covers secured by means 
of rubber bands. This mode of procedure is simpler than 
the plates and is equally as efficacious. The ^' dilutions " 
of the culture must, of course, be made in the same way 
as above described. 

Esmarch introduced a method which entirely does away 
with plates or dishes. After the test-tubes have been 
inoculated, each one is covered with an india-rubber cap 
over the plug of cotton-wool. It is then held almost 
horizontally under a running cold water tap, the upper end 
being slightly raised. The tube is now rotated on its long 
axis, and the gelatine thus solidifies round the sides of the 
tube. 

The colonies, when formed, are easily examined, both 
with a high and a low power, and may be removed by 
means of a platinum wire for examination or further culti- 
vation. 

Plate cultivations of agar-agar tubes are made in the 
same way, the plates being afterward placed in an in- 
cubator. 



BACTERIOLOGICAL METHODS. 389 

]\IoDE OF Using the Liquid Media. 

Tubes containing liquid media are inoculated simply by 
removing the plug and introducing a sterilized platinum 
needle carrying the organisms. 

Drop Cultures. — These are very important for study- 
ing the life-history of the organisms and for watching their 
movements. 

Glass slides excavated in the centre are required. To 
make a culture, one of the slides is cleaned and then 
sterilized by being held, depression downward, in the 
flame of a Bunsen burner. A little vaseline is painted 
round the excavation and the slide covered with a large 
basin or bell-jar. 

A cover-glass is next cleaned and sterilized. A drop of 
a liquid culture is then placed on it with a platinum looped 
needle, or a drop of sterile bouillon is deposited on it and 
inoculated from some culture. The prepared glass slide 
is now held over it, so that the drop comes exactly in the 
centre of the depression. Slight pressure is used, so that 
the cover-glass adheres to the vaseline. The preparation 
is turned over and a rim of vaseline painted round the 
glass. The culture can then be examined from day to day 
with an oil-immersion lens, using a diaphragm below the 
Abbe's condenser. 

Examination of Micro-organisms. 
Cover-glass Preparations are made thus : A clean 
cover-glass is taken and a drop of distilled water placed on 
it. A sterilized platinum wire is introduced into a culture 
tube and a minute portion of the growth removed, or a 
colony may be removed from a plate cultivation. The 
point of the needle is lowered into the drop of water, 
which is distributed over the cover-glass, carrying the 



390 MEDICAL MICROSCOPY. 

micro-organisms with it. When dry, the glass is passed 
three times through the flame of a Bunsen burner, and 
then stained according to the nature of the microbe which 
is being examined. 

Cover-glass Impressions. — These are employed to 
demonstrate the minute structure of colonies on plate 
cultivations. 

A clean, sterilized cover-glass is dropped on to a plate 
cultivation, a part being preferably selected where there 
are several typical well-formed colonies. It is gently 
pressed down, and then one side is raised with a sterilized 
needle. The - glass is next removed with sterilized for- 
ceps, allowed to dry, passed three times through the flame 
of a Bunsen burner, and stained. 

The methods employed for staining the principal patho- 
genic micro-organisms have been described in the previous 
chapters. For the best way of coloring those not men- 
tioned, text-books on bacteriology must be consulted. 

Experiments on Animals. — The usual method of 
infecting animals is by subcutaneous inoculation. 

A small patch of hair is shaved from a part of the body 
which the animal cannot easily reach with its tongue, and 
the skin washed with corrosive sublimate solution (i in 
I coo). A small incision is made with a sterilized scalpel, 
and a portion of the cultivation placed in the wound, 
which is allowed to close over it. In guinea pigs and 
rabbits the inside of the upper part of the hind leg is the 
part usually selected. 

More usually the inoculation is made in a fluid form. A 
watch glass is cleaned and sterilized by heat. A little 
normal saline solution which has been boiled for one 
minute is poured into it, and some of the material to be 
inoculated mixed with the fluid. A freshly prepared 
capillary pipette is taken, the point broken off with steril- 



BACTERIOLOGICAL METHODS. 39 1 

ized forceps, and some of the liquid drawn up into it, the 
amount varying with the nature of the substance to be 
experimented on. A small incision is made in the skin of 
the animal, prepared in the way already described. In 
guinea pigs a spot just internal to the mamma is usually 
chosen. The extremity of the pipette is introduced into 
the subcutaneous cellular tissue and the fluid blown out 
of the tube. The animal must be firmly held by an 
assistant. 

Another method of inoculating fluids is by means of a 
syringe. Koch's instrument is the most convenient, as it 
can be so easily washed and sterilized. It consists of a 
graduated glass cylinder, to the lower extremity of which 
a hollow needle is attached, and to the upper end an india- 
rubber bellows provided with a stop-cock. Before and 
after use it must be washed out with distilled water which 
has been boiled for three minutes, then with absolute 
alcohol, and finally with more of the distilled water. 

The needle is introduced into the watch glass contain- 
ing the mixed culture and normal saline solution (or a cul- 
ture in a liquid medium), and drawn up into the syringe 
by squeezing the balloon and loosening it again, finally 
turning the tap. The injection is made into the subcuta- 
neous tissue or peritoneal cavity. 

Another mode of infecting animals is by feeding. For 
this purpose the animal should be fed, for at least a month 
beforehand, on food which has been rendered sterile by 
boiling, and the pure cultivation or other infective material 
is then intimately mixed with the sterile food. This 
method is only applicable to certain micro-organisms, as 
most bacteria are destroyed by the action of acids of the 
stomach. 

The animal may also be placed in a closed chamber, and 
the germs introduced into the atmosphere by means of a 



392 MEDICAL MICROSCOPY. 

spray-producer, the cultivation being mixed with sterilized 
water. 

All animals chosen for experiments should be as healthy 
as possible. They must be well attended to, their cages 
carefully cleaned daily, and suitable food must be given. 
They should be kept under observation at least a month 
before they are inoculated, so as to exclude as far as possi- 
ble the possibility of previous infection. 



WORKS CONSULTED. 



Only the more important works are included here, other references are incorpo- 
rated with the text. 

Beale, Lionel, *' How to Work with the Microscope." 
" " " Microscope in Medicine." 

BizzozERO ET FiRKET, '* Microscopie Clinique." 

Bristowe, " Text-book of Medicine." 

Carpenter, <* The Microscope." 

CoATES, '' Manual of Pathology." 

CoBBOLD, " Entozoa." 

CoLMAN, " Section Cutting and Staining." 

Crookshank, " Manual of Bacteriology." 

Davis, G. S., " Practical Microscopy." 

Fagge, '* Principles and Practice of Medicine." 

Frankel, " Pathologic und Therapie der Krankheiten des Respirations 
Apparates." 

Fried LANDER, *' Microscopische Technik." 

GiBBES, Heneage, " Practical Physiology and Pathology." 

Green, *' Text-book of Pathology." 

Hamilton, " Text- book of Pathology." 

Harris and Power, *' Physiological Laboratory." 

Hassall, " Food and its Adulteration." 

Von Jaksch, " Klinische Diagnostik " (English translation by Cagney). 

"Journal of Microscopical Society." 

KuHNE, " Practical Guide to the Demonstration of Bacteria in Animal 
Tissues" (Translation by Dr. Vincent Harris). 

Lee, " Microtomist's Vade Mecum." 

Leuckart, *' Parasites of Man." 

Macdonald, J. I., " Guide to the Microscopical Examination of Drink- 
ing Water." 

Mackenzie, Hunter, " Examination of Sputum." 

Morris, " Diseases of the Kidney." 

34 393 



394 WORKS CONSULTED. 

MoQLLiN, Mansell, " Surgery." 

NoTHNAGEL, " Beitrage zur Physiologic und Pathologic dcs Darmes " 
Orth, " Compendium der Diagnostik." 
Parkes, ^' Hygiene and Public Health." 
Pfluger's " Archiv." 

Powell, Douglas, '• Diseases of the Lungs and Pleurae." 
Quain, " Dictionary of Medicine." 
Ralfe, " Diseases of the Kidney." 
Roberts, F. T., " Text-book of Medicine." 
Roberts, Sir W., " Diseases of the Kidney." 
ScHAFER, " Practical Histology." 
Stirling, *' Outlines of Practical Histology." 
*' Transactions of Pathological Society." 
'' Transactions of Royal Medical and Chirurgical Society." 
Troupe, '* Examination of Sputum." 
Ultzmann, " Impotence and Sterility." 
ViRCHOW, ''Archiv." 
Walsh, " Diseases of the Lungs." 
WoODHEAD, " Practical Pathology." 

Wynter and Wethered, *' Manual of Clinical and Practical Pathol- 
ogy-" 
ZiEGLER, '' Pathologische Anatomic." 



INDEX. 



A. 

Abbe's camera- lucida, 28 

condenser, 23 
Aberration, chromatic, 16 

spherical, 15 
Abortion, membranes of, 318 
Absolute alcohol, 45, 116 
Acarus farinae, 363 

folliculorum, 353 

scabiei, 352 
Accidental ingredients of sputum, 

277 
Acetate of potassium, 114 
Achorion Schonleinii, 350 
Acidulated alcohol, 82 
Actinomyces, 303 

in sputum, 290 

staining, 106 

Plaut's method, 106 
Adenoid tissue, 147 
Adenoma, 162 
Adeno- sarcoma, 161 
Adipose tissue, 147 
Adjustments, 11, 12 
Agar-agar, 379 

glycerine, 379 
Alcohol, absolute, 45, 116 

acidulated, S2 

dilute, 141 
Alveolar sarcoma, 160 
Ammonia, urate of, 213 
Ammonium chrom.ate, 49 
Amoeba coli, 234 
Amoebic dysentery, 235 
Amorphous phosphates in urine, 

197, 212 
Amyloid casts, 185 

reaction, 1 07 



Amyloid substance in semen, 314 
Anchylostoma duodenale, 246 
Angle of aperture, 16 
Aniline blue-black, 132 
Anguillula intestinalis, 247 
Animal parasites in vomit, 298 
Animals, experiments on, 390 
Anthrax bacillus, 304 
Aqueous humor, 140 
Areolar tissue, 146 
Arrowroot, 360, 361 
Arteries, 150 

Ascaris lumbricoides, 245 
in urine, 195 
mistax, 245 
Aspermatism, 314 
Aspergillus fumigatus, 293 
Azoospermism, 314 



B. 

Babes' incubator, 373 
Bacillus of anthrax, 304, 340 

of cholera, 230 
nostras, 231 

of glanders, 306, 341 

of leprosy, 304 

of syphilis, 305 

of tetanus, 341 

of tubercle, 103, 190, 281, 
303^ 316, 340 

of typhoid fever, 230, 341 

subtilis, 234 
Bacterium termo, 234 
Bacteriuria, 193 
Barley, 356 

Basic magnesium phosphate, 212 
Beans, 359 



395 



39^ 



INDEX. 



Bench, arrangement of, 30 
Bergamot oil, 119 
Bichromate of ammonia, 49 

of potash, 48, 141 
Bilharzia haematobia, 345 

in urine, 194 
Bilirubin, 209 
Bismarck brown, 87 
Bladder, 152 
Blood, anthrax bacilli, 340 

bilharzia haematobia, 345 

casts, 182 

corpuscles in faeces, 227 
in sputum, 261 
in urine, 173 
in vomit, 297 

counting corpuscles, 323 

estimation of haemoglobin, 

filaria sanguinis hominis, 344 
from bladder, 176 

kidney, 175 

pelvis of kidney, 176 

ureter, 176 

urethra, 176 
glanders bacilli, 341 
hsematozoa, 342 
methods of examination, 322 
plates, 322 
red corpuscles, 321 
serum, liquid, 381 

sterilized, 379 
spirillum of relapsing fever, 

341 

stains, 346 

tests for, 173 

tetanus bacilH, 341 

tubercle bacilli, 340 

typhoid bacilli, 341 

vessels, 150 

white corpuscles, 322 
Bone, 148 

Bothriocephalus latus, 241 
Bouillon, 381 
Bread, 361 

Breaking old specimens, 120 
Bronchial casts, 256 

stolons, 256 

stones, 276 



Bulk, staining in, 85 
Bunt, 362 



C. 

Cajeput oil, 119 
Calandra granaria, 363 
Calculi, 214 
Camera-lucida, 19 

Abbe's, 2S 

simple prismatic, 28 

use of, 27 
Canada balsam, 115, 119 
Cancer cells in urine, 189 

in uterine discharge, 316 
Capillaries, 150 
Capsules, 40 

Carbonate of lime in urine, 212 
Carcinomata, 163 
Carmine, alum, 84 
Carmine ammonium, 84 

and picric acid, 80, 123 

borax, 85 

injection mass, 135 

lithium, 81 
Cartilage, 147 

elastic, 148 

fibro-, 148 

hyaline, 147 

ossifying, 148 
Castor oil, 115 
Casts, 180, et seq. 

amyloid, 185 

blood, 182 

epithelial, 183 

false, 187 

fatty, 186 

granular, 184 

hyaline, 186 

micrococci, 184 

pus, 182 

seminal tubes, 188 
Cathcart's microtome, 31, 63 
Caustic potash solution, 141 
" Cayenne pepper " grains, 169 
Cedar wood oil, 119 
Celloidin, cutting in, 66, 123 

embedding in, 58 



INDEX. 



397 



Cementing materials, 115 
Central nervous system, cutting, 
126 
hardening, 125 
staining, 128 
Cercomonas intestinalis, 236 
Charcot- Leyden crystals in faeces, 
227 

in sputum, 274 
Chicory, 364 
Cholesterin, 213 

in sputum, 275 
Chromatic aberration, 16 
Chromic acid, 48 

and spirit, 49 
Chylous exudations, 301 
Ciliated epithelium, 146, 262 
Clearing, 1 17 
Clips, 25 

Cloves, oil of, 118 
Cocoa, 366 
Coffee, 364 
Cohn's fluid, 382 
Cohnheim's gold method, 95 
Cold injection, 135 
Collodionization of sections, 69 
Colloid cancer, 166 
Colonies, separation of, 387 
Columnar epithelium, 145, 262 
Compound granule cells, 260 
Concretions in sputum, 276 

intestinal, 222 
Condenser, ii 

Abbe's, 24 
Constant pressure apparatus, 137 
Corpora amylacea, 271 
Corrosive sublimate, 50 
Cotton fibres, 279 
Cover-glass impressions, 390 

preparations, 389 
Cover-glasses, 39 
Creosote, 118 
Crystals in faeces, 226 

in sputum, 274 
Cubical epithelium, 262 
Cultivating dishes, 373 
Cultivating media, liquid, 380 

solid, 376 
Cultivations, potato, 376 



Cultivations, test-tube, 382 

plate, 384 
Cultures, drop, 389 
Curschmann's spirals, 268 
Cutting embedded sections, 67, 70 
Cutting frozen sections, 63, 65, 66 
Cutting paraffin sections, 70 
Cutting sections, 62, et seq. 
Cylindrical- celled epitheliomata, 

163 
Cylindroids, 187 
Cysticercus cellulosae, 307 
Cystin, 205 
Cysts, dermoid, 311 

hydatid, 308 

ovarian, 310 



Dammar varnish, 115, 120 

Damp chamber, 376, 386 

Decalcifying, 52 

Decidua, 319 

Deep growths, 382 

Defining power, 15 

Dermoid cysts, 311 

Dextrin, solution of, 56 

Diaphragms, II, 14 

Diphtheritic membrane, 272 

Dissociation, 141 

Distoma haematobium in blood, 345 

in urine, 194 
Distoma hepaticum, 243 

lanceolatum, 243 

sinense, 243 
Drawing objects, 27 
Drinking water, 366 
Drop cultures, 389 
Dysmenorrhoea,membranes of, 318 



E. 

Ear, 150 
Ear-cockle, 363 
Echinococci, 308 
in urine, 195 
Echinococcus membrane in spu- 
tum, 273 



393 



INDEX. 



Eczema marginatum, 350 
Ehrlich's method for tubercle 

bacilli, 286 
Elastic cartilage, 148 
fibres in vomit, 295 
tissue, 146, 263 
Elastic tissue, characters of, 265 
Fenwick's method, 264 
from larynx, 265 
from lung, 265 
from gangrene, 267 
in phthisis, 266 
in pneumonia, 266 
mode of examining, 263 
signification, 266 
with lime salts, 267 
Embedding, 55, etseq. 
in celloidin, 58 
in paraffin, 56, 60 
L's, 61 
simple, 56 
tray, 56 
Embryological specimens, 152 
Embryonal tissue, 147 
Enchondromata, 155 
Enteritis mucosa, 221 
Entozoa in sputum, 294 
Eosin, 79 

Epithelial casts, 183 
Epitheliomata, 163 
Epithelium, bladder, 171 
ciliated, 145, 262 
columnar, 145, 262 
cubical, 262 
in f?eces, 228 
in semen, 314 
in sputum, 262 
in urine, 170 
in vomit, 296 
male urethra, 170 
pelvis of kidney, 171 
renal, 172 
secretory, 145 
squamous, 144, 262 
transitional, 146 
ureter, 171 
vaginal, 170 
Ergot, 362 
Erlicki's solution, 48 



Erysipelas, urine in, 192 
Esmarch's method of cultivation, 

388 

Eustrongylus gigas, 195 
Experiments on animals, 390 
Extraneous matters in urine, 215 
Exudations, chylous, 301 

hemorrhagic, 300 

purulent, 301 

putrid, 301 

sero-purulent, 301 

serous, 299 
Eye, 150 
Eye-pieces, 11, 19 



F. 

Faeces, areolar tissue, 225 
amoeba coli, 234 
bacillus of cholera, 230 

of cholera nostras, 231 

of tubercle, 229 

of typhoid fever, 230 
Charcot-Leyden crystals, 227 
color, 218 
consistence, 218 
elastic fibres, 225 
epithelium, 228 
examination of, 218 
fat in, 226 
foreign bodies, 221 
green, 220 
haematoidin, 227 
in cholera, 219 
in dysentery, 219 
in health, 225 
in jaundice, 219 
in pancreatic obstruction, 219 
in psilosis, 219 
in typhoid fever, 219 
infusoria, 236 
leucocytes, 228 
moulds, 233 
mucous and fibrinous shreds, 

221 
muscular fibres, 225 
non-pathogenic organisms, 
232 



INDEX. 



399 



Fseces, odor, 220 

oxalate of lime, 227 

protozoa, 234 

pus, 228 

quantity, 217 

reaction, 220 

red blood-corpuscles, 227 

starch granules, 226 

vegetable cells, 226 
Fallopian tubes, 152 
False casts, 187 
Farrant's solution, 1 14 
Fat in faeces, 226 

in urine, 180 
Fatty casts, 186 

crystals in sputum, 274, 275 
Fenwick's method for elastic tis- 
sue, 264 
Fibrinous clots, 317 
Fibro cartilage, 148 
Fibromata, 153 
Filaria sanguinis hominis, 344 

in urine, 194 
Filters, 40 
Flasks, 373 
Flatness of field, 16 
Fleischl's haemometer, 332 
Flemming's method for karyoky- 

nesis, 108 
Flour, 362 

Fluid mounting media, no 
Food, examination of, 354, et seq. 
Forceps, 40 

Foreign bodies in faeces, 221 
in sputum, 277 
in urine, 214 
Freezing media, 56 
Fresh tissues, 139 
Friedlander's pneumococci, 288 



Gabbet's method, 287 
Gall-stones, 223 
Gelatine, nutrient, 377 
Gelatinous tissue, 147 
General stains, 87 
Gibbes' double stain, 105 



Gibbes' method for tubercle bacilli, 

288 
Glanders, bacillus of, 306 
Glass rods, 40 

slides, 38 
Glycerine, 112 

agar-agar, 379 

and alcohol, 113 

jelly, 113 

mounting fluid, 114 
Gold chloride, 94 
Gonococcus, 306 
Gowers' haemocytometer, 323 

haemoglobinometer, 330 
Gram's method, loi 

Weigert's modification 
of, 102 

solution, 41, 1 01 
Granular casts, 184 
Gravel, 214 

Gulland's method of cutting, 73 
Gum and dextrin, 56 

and syrup, 56 



H. 

Haematoidin in faeces, 227 

in sputum, 273 

in urine, 209 
Haematoxyhc eosin, 80 
Haematoxyhn, 77 

and eosin, 77, 123 
Haematozoa of malaria, 342 
Haematuria, 175 
Haemocytometer, Gowers', 323 

Thoma-Zeiss, 326 
Haemoglobinometer, 330 
Haemoglobinuria, 176 
Haemometer, 332 
Haemoptysis, 251 

spurious, 254 
Hardening, 42, et seq. 

choice of reagent, 43 
Hemorrhagic exudations, 300 
Hippuric acid, 208 
Hofmann's solution, 347 
Hot-air sterilizer, 371 
Hot-water filter, 372 



400 



INDEX. 



Hyaline cartilage, 147 

casts, 186 
Hydatid cysts, 308 
Hydrochloric acid, 53 
Hydronephrosis, 311 



Illumination, 22 

artificial, 25 
Incubator, 373 
Indian corn, 357 
Indigo, 213 

Infiltration methods, 58 
Infusoria in feces, 236 ' 

in sputum, 293 

in urine, 196 
Injection of tissues, 135 
Intestinal concretions, 222 
Intestines, 151 
Intussusception, 222 
Iodine green, 86, 107 

in iodide of potassium, 41 

mounting fluid, 114 
Iodized serum, 140, 141 
Iron reaction, 108 
Itch mite, 352 



Jolgi's sublimate method, 133 



K. 

Karyokinetic figures, 108 
Kidney, 151 

Kleinenberg's solution, 51 
Koch's steam sterilizer, 371 
Kiihne's method, 98 



L. 

Lamp, 25 

Landois' fluid, 142 
Lens, oil immersion, 23 
Leprosy bacillus, 304 
Leptothrix buccalis, 292 



Leucaemia, 335 
Leucin, 208 

in sputum, 276 
Leucocytes, 335 

in faeces, 228 

in sputum, 259 

in urine, 176 

in vomit, 297 
Leucocyth^mia, 335 
Leucocytosis, 337 
Leveling apparatus, 386 
Lientery, 221 
Lime, carbonate of, 212 

neutral phosphate of, 211 

oxalate of, 198, 202 

sulphate of, 204 
Linen fibres, 279 
Liver, 151 

Loffler's solution, 97 
Lungs, 151 
Lung stones, 276 
Lymphatic glands, 150 
Lympho-sarcoma, 1 61 



M. 

Magenta, 168 
Magnesium phosphate, 212 
Maize, 357 
Malaria, 342 
Marsh's cement, 115 
Mast-zellen, 100. 
Medullary cancer, 165 
Megalob lasts, 339 
Mefena, 219 
Melanaemia, 339 
Melanotic carcinoma, 166 

sarcoma, 160, 1 61 
Membrane in vomit, 297 
Membranes, examination of, 140 

of dysmenorrhoea, 3 1 8 

of early abortion, 318 
Methyl anihne violet, loi, 107 
Methylated spirit, 46 
Methylene blue, 96 
Micrococci, casts composed of, 184 
Micrococcus tetragonus, 291 

ureae, 196 



INDEX. 



401 



Microcytes, ^^S 
Microcythaemia, 337 
Micro-organisms, 369 

in vomit, 2^8 
Micrometer, eye-piece, 29 

stage, 29 
Microscope, base of, 1 1 

choice of, 9 

column of, 1 1, 12 

compound, 10 

makers of, 19, 20 

optics of, 10 

requisites of a good, 1 2 

simple, 9 

stage of, II, 12 

stand of, II 

use of, 20 
Microsporon furfur, 350 
Microtome, Cathcart's, 31 

Reichert's, 35, 67 

rocking, 36 

Swift's, 33 

Williams' ether, 34 

WiUiams' ice and salt, 35 
Mirror, li 
Morbid growths, fragments of, in 

urine, 189 
Moulds in faeces, 233 

in urine, 196 
Mounting, methods of, 116, 117 
Mucous and fibrinous shreds in 
faeces, 221 
cancer, 166 
Mucus corpuscles, 259 

in urine, 179 

simulating membranes, 317 
Miiller's fluid, 46 
Multiple staining, 8S 
Muscle fibre, 295 

in faeces, 225 
Myelin drops, 260 
Myeloid sarcoma, 160 
Myo-fibromata, 157 
Myxomata, 154 
Myxo-sarcoma, 160 

N. 

Needles, 40 

N§elsen-Ziehl method, 103, 282 



Nerve-cells, 149 

endings, 149 

fibres, 149 
Neutral phosphate of lime, 211 
Nitrate of silver, 93 
Nitric acid, 52 
Non-pathogenic organisms in 

faeces, 232 
Normal saline solution, 41, 139 
Nose-piece, 18 

use of, 25 
Nummulated sputa, 255 
Nutrient agar-agar, 379 

gelatine, 377 

O. 

Oatmeal, 357, 364 
Objectives, ii 

qualities of good, 14 

selection of, 14 
Oculars, 19 
QEsophagus, 151 
Oidium albicans, 292 

arbortifaciens, 362 
Oil immersion lens, 23 

of bergamot, 119 

of cajeput, 119 

of cedar wood, I19 

of cloves, 118 

of origanum, 119 
Oligocythaemia, 334 
Olizoospermism, 314 
Organized urinary deposits, 170 
Origanum, oil of, 119 
Orseille, 88 
Osmic acid (hardening), 51 

(staining), 96 
Ossifying cartilage, 148 
Osteomata, 156 
Ovarian cysts, 310 
Ovaries, 152 

Oxalate of lime in faeces, 227 
in sputum, 276 
in urine, 198, 202 
Oxyuris vermicularis, 245 



Pacinian corpuscles, 149 
Pal-Exner method, 132 



402 



INDEX. 



Pal's method, 131 
Pancreas, 151 
Papillomata, 161 
Paraffin, cutting in, 70, 72 
embedding in, 56, 60 
Paramaecium coli, 236 
Pasteur's fluid, 381 
Peas, 359 

Pediculus capitis, 351 
pubis, 352 
vestimenti, 352 
Penetration power of lenses, 16 
Penicillium glaucum, 196 
Perenzi's solution, 53 
Phosphates, amorphous, 197 
in urine, 197, 205 

triple, 197, 211 
magnesium, 212 
of lime, amorphous, 212 
neutral, 211 
Picric acid (decalcifying), 53 
(hardening), 51 
(staining), 82 
Picro carmine, 82 
Pityriasis versicolor, 350 
Plastic bronchitis, 256 
Platinum chloride, 50 

needles, 375 
Plaut's method for actinomyces, 

106 
Pneumococci, Frankel's, 290 

Friedlander's, 288 
Poikilocytes, 339 
Poikilocytosis, 339 
Polycythaemia, 335 
Potassium bichromate, 48, 141 
Potato, 360 

cultivation, 376 
knife, 374 
Protozoa, in fceces, 234 
Puccinia graminis, 362 
Purulent exudations, 301 
actinomyces, 303 
staphylococcus pyogenes 

albus, 303 
staphylococcus pyogenes 

aureus, 302 
tubercle bacilli, 303 
Pus casts, 182 



Pus in faeces, 228 

in sputum, 259 

casts in urine, 178 

from bladder, 179 

from kidney, 178 

from pelvis of kidney, 178 

from urethra, 179 
Putrid exudations, 301 
Pyelitis, 178 



Ranvier's gold method, 95 

Reagents, 40 

Recurrent fever, urine in, 192 

Reichert's microtome, 35, 67 

Relapsing fever, 341 

Renal epithelium, 172 

Renaut's haematoxylic eosin, 80 

Resolving power of lenses, 16 

Rice, 358 

Ringworm, 348 

Rye, 357 

Rocking microtome, 36, 72 

Round worm, 245 

Round-celled sarcoma, 158 

Rubin, 87 

Rust, 362 



S. 

Sable intestinal, 224 
Saccharomyces cerevisise in urine, 

196 
Saccharomycetes in f^ces, 233 
Safranin, 86 
Sago, 359 
Saline solution, 41 
Salivary corpuscles, 260 
Salkouski's fluid, 167 
Salt solution, 141 
Sarcinae in urine, 194 

in vomit, 298 

pulmonis, 293 
Sarcomata, 158 
Sarcoptes hominis, 352 
Scabies, 352 
Schyzomycetes in faeces, 233 



INDEX. 



403 



Scirrhus, 164 
Scybala, 222 
Sebaceous glands, 150 
Secretory epithelium, 145 
Section lifter, 40 
Sections, cutting, 62, et seq. 
Selective stains, 92 
Seminal cells, 314 

fluid, 312 

granules, 314 

tubes, casts of, 188 
Septic micro organisms in sputum, 
291 
in urine, 192 
Series cutting, 70 
Sero-purulent exudations, 301 
Serous fluid, 139 

exudations, 299 
Silk- fibres, 279 
Silver nitrate, 93 
Smut, 362 
Soaps of lime in urine, 210 

of magnesia in urine, 210 
wSoda, urate of, 202 
Softening tissues, 141 
Solidifying mounting media, 116 
Spermatic crystals, 315 

stains, 315 
Spermatocele, 311 
Spermatozoa, 188, 313 
Spherical aberration, 15 
Spheroidal- celled cancer, 164 
Spindle-celled sarcoma, 160 
Spirillum of relapsing fever, 341 
Spleen, 151 

Spurious haemoptysis, 254 
Sputum,accidental ingredients, 277 

actinomyces, 290 

aspergillus fumigatus, 293 

black, 253 

blood in, 251 

bronchial casts, 256 

brown, 252 

Charcot-Leyden crystals, 274 

chocolate, 252 

cholesterin crystals, 275 

collection of, 249 

color, 251 

compound granular cells, 260 



Sputum, concretions, 276 
connective tissue, 268 
consistence, 250 
corpora amylacea, 271 
Curschmann's spirals, 268 
dark red, 254 

diphtheritic membrane, 272 
echinococcus membrane, 273 
elastic tissue, 263 
entozoa, 294 
epithelium, 262 
fatty crystals, 275 
foreign bodies, 279 
fungi, 292 
green, 252, 253 
haematoidin, 273 
in abscess of liver, 25 3 

of lung, 250 
in asthma, 269 
in bronchiectasis, 250, 256 
in bronchitis, 250 
in brown induration of lungs, 

252 
in empyema, 255 
in gangrene of lung, 252, 256 
in haemoptysis, 251 
in malignant disease, 254 
in oedema of lungs, 250 
in phthisis, 250 
in pneumonia, 250, 253 
infusoria, 293 
lemon-colored, 252 
leptothrix buccalis, 292 
leucin, 276 
leucocytes, 259 
micrococcus tetragonus, 291 
mucus corpuscles, 259 
myelin drops, 260 
nummulated, 255 
odor, 255 

Oidium albicans, 292 
oxalate of lime, 276 
plastic bronchitis, 256 
pneumococci, 288 
pus cells, 260 
quantity, 249 
red blood cells, 261 
reddish-brown, 252 
rust-coloredj 252 



404 



INDEX. 



Sputum, salivary corpuscles, 260 

sarcinae pulmonis, 293 

septic micro-organisms, 291 

stratification, 255 

tonsillar casts, 270 

transparency, 251 

triple phosphate, 276 

tubercle bacilli, 281 

tyrosin, 276 

varieties of, 249 

yellow, 253 
Squamous-celled epitheliomata, 

163 

Squamous cells in sputum, 262 
in urine, 170 

epithelium, 144, 262 
Staining, 74, et seq. 

in bulk, 85 

multiple, ^% 

treble, 89 
Stains, choice of, 77 

classification of^ 74, 75 

general, 87 

management of, 76 

selective, 92 
Staphylococcus pyogenes albus, 

303 

aureus, 302 
Starch, arrowroot, 360, 361 

barley, 356 

bean, 359 

maize, 357 

oatmeal, 357 

pea, 359 

potato, 360 

rice, 358 

rye, 357 

sago, 359 

tapioca, 359 

wheat, 355 

granules in faeces, 226 
in vomit, 296 
Steam sterilizer, 370 
Sterilized blood serum, 379 
Sterilizer, hot-air, 371 

steam, 370 
Stools (see Faeces). 
Stomach, 15 1 
Striped muscle, 148 



wSub-stage, li 
Surface-growths, 388 
Sweat glands, 150 
Swift's microtome, 32, 65 
Syphilis, bacillus of, 305 



Tactile cells, 149 
T^nia flavopuncta, 242 

leptocephala, 242 

madagascariensis, 242 

mediocanellata, 237 

nana, 241 

saginata, 237 

solium, 239 
Tapioca, 359 
Tea, 364 
Teasing, 140 
Teeth, 148 
Testicle, 152 
Tinea circinata, 350 

marginata, 350 

tonsurans, 350 

versicolor, 350 
Thoma-Zeiss haemocytometer, 326 
Thread- worm, 245 
Thymus gland, 151 
Thyroid gland, 15 1 
Tongue, 150 
Tonsillar casts, 270 
Transitional epithelium, 146 
Treble staining, 89 
Trichina spiralis, 247, 307 
Trichocephalus dispar, 246 
Trichomonas intestinalis, 236 

vaginalis, 316 
Trichophyton tonsurans, 348 
Triple phosphates in sputum, 276 

in urine, 197, 210 
Tubercle bacilli in blood, 340 
in discharges, 303 
in f^ces, 229 
in sputum, 281 
in urine, 190 
in vaginal discharges,3i6 
staining, 103 
Turmeric, 360 



INDEX. 



405 



Turpentine, oil of, 119 
Tyrosin, 207 

in sputum, 276 



U. 

Unstriped muscle, 148 
Uredo foetida, 362 
segetum, 362 
Urate of ammonia, 213 

of soda, 202 
Urates, 197, 200 
Uric acid, 198 

Urinary sediments, 167, et seq. 
organized, 169 
non- organized, 196 
preservation of, 216 
Urine, amorphous phosphates of 
lime, 212 
phosphates, 197 
urates, 197, 200 
amyloid casts, 185 
ascaris lumbricoides, 195 
bacteriuria, 193 
basic magnesium phosphate, 

212 
bilharzia haematobia, 194 
bilirubin, 209 
bladder epithelium, 171 
blood-casts, 182 
cancer cells, 189 
carbonate of lime, 212 
casts, 180 

composed of crystals, 187 
of micrococci, 184 
of urates, 187 
of seminal tubes, 188 
cells from kidney, 171 
pelvis of kidney, 171 
ureter, 171 
cholesterin, 213 
cylindroids, 187 
cystin, 205 

distoma haematobium, 194 
echinococci, 195 
epithelial casts, 183 
epithelium from male urethra, 
170 



Urine, epithelium in, 170 
erysipelas, 192 
eustrongylus gigas, 195 
extraneous matters, 215 
false casts, 187 
fat in, 180 
fatty casts, 186 
filaria sanguinis hominis, 194 
granular casts, 184 
gravel, 214 
hsematoidin, 209 
hippuric acid, 208 
hyaline casts, 186 
indigo, 213 
in dyspepsia, 202 
infusoria, 196 
in gout, 200 
in gonorrhoea, 171 
in haemoglobinuria, 176 
in nocturnal emissions, 171 
leucin, 208 
leucocytes, 176 
method of examining, 167 
morbid growths, 189 
moulds, 196 
mucus, 179 

neutral phosphate of lime, 21 1 
non-pathogenic organisms, 

196 
nuclei of epithelial cells, 174 
oxalate of lime, 198, 202 
parasites, 190 
penicillium glaucum, 196 
phosphates, 197, 205 
pus casts, 182 
cells, 176 
red blood corpuscles, 173 
renal epithelium, 172 
saccharomyces cerevisise, 196 
sarcinae, 194 

septic micro-organisms, 192 
soaps of lime and magnesia, 

210 
source of blood in, 175 
spirmatozoa, 188 
spirillum of recurrent fever, 

192 
sporules of fungi, 174 
squamous cells in, 170 



4o6 



INDEX. 



Urine, sulphate of lime, 204 
test for blood, 173 
triple phosphates, 210 
tubercle bacilli, 190 
tyrosin, 207 
urate of ammonia, 213 

of soda, 202 
uric acid, 197, 198 
vaginal cells in, 170 
vibriones, 196 
xanthin, 206 

Uterus, 152 



Vaginal casts, 318 

secretion, 316 
Valentin's knife, 36, 142 
Vegetable cells in faeces, 226 
Veins, 150 
Vesuvin, 87 
Vibrio tritici, 363 
Vomit, animal parasites, 298 

blood cells, 297 

elastic fibres, 296 

epithelium, 296 

food matter, 295 

leucocytes, 297 

membranes, 297 

method of examining, 295 

micro-organisms, 298 

muscle fibres, 295 

sarcinae, 298 

starch granules, 296 

vegetable fibres, 296 



Vomit, vegetable parasites, 29 

yeasts, 298 
Von Ebner's solution, 53 



W. 

Watch glasses, 40 
Water, examination of, 366 
Webb's solution, 56 
Weevil, 363 

Weigert's method fornervous sys- 
tem, 129 
modification of Gram's meth- 
od, 102 
Wheat, 355 
Whipworm, 246 
White fibrous tissue, 35 
Williams' ice and salt microtome, 

35.66 

Woolen fibres, 280 



Xanthin, 206 
Xylol, 119 



X. 



Y. 



Yeasts in vomit, 298 



Ziehl's method, 103, 282 



CATALOGUE No. 7. 



JULY, 1892. 



A CATALOGUE 

OF 

BOOKS FOR Students. 

INCLUDING THE 

? QUIZ-COMPENDS ? 





CONTENTS. 




PAGE 


PAGE 


New Series of Manuals. 2,^a,<> 


Obstetrics lo 


Anatomy, 


. .6 


Pathology, Histology, . . ii 


Biology, 


. II 


Pharmacy, . . . . 12 


Chemistry, . 


. 6 


Physical Diagnosis, . .11 


Children's Diseases, 


. 7 


Physiology, . . . . n 


Dentistry, . 


, 8 


Practice of Medicine, . 11, 12 


Dictionaries, 


8, i6 


Prescription Books, . . 12 


Eye Diseases, 
Electricity, . 


. 8 


?Quiz-Compends? . 14,15 


• 9 


Skin Diseases, . . .12 


Gynaecology, 


. lO 


Surgery and Bandaging, . 13 


Hygiene, 


• 9 


Therapeutics, . . .9 


Materia Medica, . 


• 9 


Urine and Urinary Organs, 13 


Medical Jurisprudence 


• 9 


Venereal Diseases, . . 13 


Nervous Diseases, 


. lO 






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''Walsham, besides being an excellent surgeon, is a teacher in 
its best sense, and having had very great experience in the 
preparation of candidates for examination, and their subsequent 
professional career, may be relied upon to have carried out his 
work successfully. Without following out in detail his arrange- 
ment, which is excellent, we can at once say that his book is an 
embodiment of modern ideas neatly strung together, with an amount 
of careful organization well suited to the candidate, and, indeed, to 
the practitioner.*' — British Medical Journal, 

Price of each Book, Cloth, $3.00 ; Leather, $3.60. 



THE NEW SERIES OF MANUALS. 8 

No. 2. DISEASES OP "WOMEN. 150 Illus. 

NEW EDITION. 

The Diseases of Women. Including Diseases of the 
Bladder and Urethra. By Dr. F. Winckel, Professor 
of Gynaecology and Director of the Royal University 
Clinic for Women, in Munich. Second Edition. Re- 
vised and Edited by Theophilus Parvin, m.d., 
Professor of Obstetrics and Diseases of Women and 
Children in Jefferson Medical College. 150 Engrav- 
ings, most of which are original. 
" The book will be a valuable one to physicians, and a safe and 

satisfactory one to put into the hands of students. It is issued in a 

neat and attractive form, and at a very reasonable price." — Boston 

Medical and Surgical Journal . 

No. 3. OBSTETRICS. 227 Illustrations. 
A Manual of Midwifery. By Alfred Lewis Galabin, 
M.A., M.D., Obstetric Physician and Lecturer on Mid- 
wifery and the Diseases of Women at Guy's Hospital, 
London; Examiner in Midwifery to the Conjoint 
Examining Board of England, etc. With 227 Illus. 
** This manual is one we can strongly recommend to all who 
desire to study the science as well as the practice of midwifery. 
Students at the present time not only are expected to know the 
principles of diagnosis, and the treatment of the various emergen- 
cies and complications that occur in the practice of midwifery, but 
find that the tendency is for examiners to ask more questions 
relating to the science of the subject than was the custom a few 
years ago. * * * The general standard of the manual is high ; 
and wherever the science and practice of midwifery are well taught 
it will be regarded as one of the most important text-books on the 
subject." — London Practitioner. 

No. 4. PHYSIOLOGY. Fifth Edition. 

321 ILLUSTRATIONS AND A GLOSSARY. 
A Manual of Physiology. By Gerald F. Yeo, m.d., 

F.R.C.S., Professor of Physiology in King's College, 

London. 321 Illustrations and a Glossary of Terms. 

Fifth American from last English Edition, revised and 

improved. 758 pages. 

This volume was specially prepared to furnish students with a 
new text-book of Physiology, elementary so far as to avoid theories 
which have not borne the test of time and such details of methods 
as are unnecessary for students in our medical colleges. 

** The brief examination I have given it was so favorable that I 
placed it in the list of text-books recommended in the circular of the 
University Medical College." — Prof. Lewis A. Stimson, m.d., 
57 East 33d Street, New York. 

Price of each Book, Cloth, $3.00; Leather, $3.50. 



THE NEW SERIES OF MANUALS. 



No. 6. DISEASES OP CHILDREN. 

SECOND EDITION. 

A Manual. By J. F. Goodhart, m.d., Phys. to the 
Evelina Hospital for Children ; Asst. Phys. to 
Guy's Hospital, London. Second American Edition. 
Edited and Rearranged by Louis Starr, m.d., Clinical 
Prof, of Dis. of Children in the Hospital of the Univ. 
of Pennsylvania, and Physician to the Children's Hos- 
pital, Phila. Containing many new Prescriptions, a list 
of over 50 Formulae, conforming to the U. S. Pharma- 
copoeia, and Directions for making Artificial Human 
Milk, for the Artificial Digestion of Milk, etc. Illus. 

** The merits of the book are many. Aside from the praiseworthy 
work of the printer and binder, which gives us a print and page 
that delights the eye, there is the added charm of a style of writ- 
ing that is not wearisome, that makes its statements clearly and 
forcibly, and that knows when to stop when it has said enough. 
The insertion of typical temperature charts certainly enhances the 
value of the book. It is rare, too, to find in any text-book so many 
topics treated of. All the rarer and out-of-the-way diseases are 
given consideration. This we commend. It makes the work 
valuable." — Archives of Pedriatics , July , i8go. 

" The author has avoided the not uncommon error of writing a 
book on general medicine and labeling it * Diseases of Children,' 
but has steadily kept in view the diseases which seemed to be 
incidental to childhood, or such points in disease as appear to be so 
peculiar to or pronounced in children as to justify insistence upon 
them. * * * A safe and reliable guide, and in many ways 
admirably adapted to the wants of the student and practitioner." — 
American Journal of Medical Science. 

'* Thoroughly individual, original and earnest, the work evi- 
dently of a close observer and an independent thinker, this book, 
though small, as a handbook or compendium is by no means made 
up of bare outlines or standard facts." — The Therapeutic Ga- 
zette. 

" As it is said of some men, so it might be said of some books, 
that they are ' born to greatness.' This new volume has, we 
believe, a mission, particularly in the hands of the younger 
members of the profession. In these days of prolixity in medical 
literature, it is refreshing to meet with an author who knows both 
what to say and when he has said it. The work of Dr. Goodhart 
(admirably conformed, by Dr. Starr, to meet American require- 
ments) is the nearest approach to clinical teaching without the 
actual presence of clinical material that we have yet seen." — /^ew 
York Medical Record, 

Price of each Book, Cloth, $3.00 ; Leather, $3.|Q, 



THE NEW SERIES OF MANUALS. 



No. 6. PRACTICAL THERAPEUTICS. 

FOURTH EDITION, WITH AN INDEX OF DISEASES. 

Practical Therapeutics, considered with reference to 
Articles of the Materia Medica. Containing, also, an 
Index of Diseases, with a list of the Medicines 
applicable as Remedies. By Edward John Waring, 
M.D., F.R.c.P. Fourth Edition. Rewritten and Re- 
vised by Dudley W. Buxton, m.d., Asst. to the Prof, 
of Medicine at University College Hospital. 

*' We wish a copy could be put in the hands of every Student or 
Practitioner in the country. In our estimation, it is the best book 
of the kind ever written." — N. V. Medical Journal. 

'* Dr. Waring's Therapeutics has long been known as one of the 
most thorough and valuable of medical works. The amount of 
actual intellectual labor it represents isammense. . . . An in- 
dex of diseases, with the remedies appropriate for their treatment, 
closes the volume." — Boston Medical and Surgical Reporter. 

" The plan of this work is an admirable one, and one well calcu- 
lated to meet the wants of busy practitioners. There is a remark- 
able amount of information, accompanied with judicious comments, 
imparted in a concise yet agreeable style." — Medical Record. 

No. 7. MEDICAL JURISPRUDENCE AND 
TOXICOLOGY. 

THIRD REVISED EDITION. 

By John J. Reese, m.d.. Professor of Medical Jurispru- 
dence and Toxicology in the University of Pennsyl- 
vania ; President of the Medical Jurisprudence Society 
of Phila. ; Third Edition, Revised and Enlarged. 

*' This admirable text-book." — Amer.Jour. of Med. Sciences. 

" We lay this volume aside, after a careful perusal of its pages, 
with the profound impression that it should be in the hands of every 

doctor and lawyer. It fully meets the wants of all students 

He has succeeded in admirably condensing into a handy volume all 
the essential points." — Cincinnati Lancet and Clinic. 

*' The book before us will, we think, be found to answer the ex- 
pectations of the student or practitioner seeking a manual of juris- 
prudence, and the call for a second edition is a flattering testimony 
to the value of the author's present effort. The medical portion 
of this volume seems to be uniformly excellent, leaving little for 
adverse criticism. The information on the subject matter treated 
has been carefully compiled, in accordance with recent knowledge. 
The toxicological portion appears specially excellent. Of that por- 
tion of the work treating of the legal relations of the practitioner 
and medical witness, we can express a generally favorable ver- 
dict." — Physician and Surgeon, Ann Arbor ^ Mich. 

Price of each Book, Cloth, $3,00; Leather, $3.50. 



6 STUDENTS' TEXT-BOOKS AND MANUALS. 

ANATOMY. 

Macalister's Human Anatomy. 8i6 Illustrations. A new 
Text-book for Students and Practitioners, Systematic and Topo- 
graphical, including the Embryology, Histology and Morphology 
of Man. With special reference to the requirements of 
Practical Surgery and Medicine. With 8i6 Illustrations, 
400 of which are original. Octavo. Cloth, 7,50; Leather, 8.50 

Ballou's Veterinary Anatomy and Physiology. Illustrated. 
By Wm. R. Ballou, m.d., Professor of Equine Anatomy at New 
York College of Veterinary Surgeons. 29 graphic Illustrations. 
i2mo. Cloth, 1. 00; Interleaved for notes, 1.25 

Holden's Anatomy. A manual of Dissection of the Human 
Body. Fifth Edition. Enlarged, with Marginal References and 
over 200 Illustrations. Octavo. 

Bound in Oilcloth, for the Dissecting Room, $4.50. 
" No student of Anatomy can take up this book without being 
pleased and instructed. Its Diagrams are original, striking and 
suggestive, giving more at a glance than pages of text description. 
* * * The text matches the illustrations in directness of prac- 
tical application and clearness of detail." — New York Medical 
Record. 

Holden's Human Osteology. Comprising a Description of the 
Bones, with Colored Delineations of the Attachments of the 
Muscles. The General and Microscopical Structure of Bone and 
its Development. With Lithographic Plates and Numerous Illus- 
trations. Seventh Edition. 8vo. Cloth, 6.00 

Holden's Landmarks, Medical and Surgical. 4th ed. Clo., 1.25 
Heath's Practical Anatomy. Sixth London Edition. 24 Col- 
ored Plates, and nearly 300 other Illustrations. Cloth, 5.00 
Potter's Compend of Anatomy. Fifth Edition. Enlarged. 
16 Lithographic Plates. 117 Illustrations. See Page 14. 

Cloth, 1. 00; Interleaved for Notes, 1.25 

CHEMISTRY. 

Hartley's Medical Chemistry. Second Edition. A text-book 
prepared specially for Medical, Pharmaceutical and Dental Stu- 
dents. With 50 Illustrations, Plate of Absorption Spectra and 
Glossary of Chemical Terms. Revised and Enlarged. Cloth, 2.50 

Trimble. Practical and Analytical Chemistry. A Course in 
Chemical Analysis, by Henry Trimble, Prof, of Analytical Chem- 
istry in the Phila. College of Pharmacy. Illustrated. Fourth 
Edition, Enlarged. 8vo. Cloth, 1.50 

9Gf See pages 2 to 3 /or list 0/ Students' Manuals . 



STUDENTS' TEXT-BOOKS AND MANUALS. 7 

Chemistry : — Continued. 

Bloxam's Chemistry, Inorganic and Organic, with Experiments. 
Seventh Edition. Enlarged and Rewritten. 281 Illustrations. 

Cloth, 4.50; Leather, 5.50 

Richter's Inorganic Chemistry. A text-book for Students. 
Third American, from Fifth German Edition. Translated by 
Prof. Edgar F. Smith, ph.d. 89 Wood Engravings and Colored 
Plate of Spectra. Cloth, 2.00 

Richter's Organic Chemistry, or Chemistry of the Carbon 
Compounds. Illustrated. Second Edition. Cloth, 4,50 

Symonds. Manual of Chemistry, for the special use of Medi- 
cal Students. By Brandreth Symonds, a.m., m.d., Asst. 
Physician Roosevelt Hospital, Out- Patient Department; Attend- 
ing Physician Northwestern Dispensary, New York. i2mo. 

Cloth, 2.00 

Leffmann's Compend of Chemistry. Inorganic and Organic. 
Including Urinary Analysis. Third Edition. Revised. 

Cloth, 1. 00; Interleaved for Notes, 1.25 

Leffmann and Beam. Progressive Exercises in Practical 
Chemistry. i2mo. Illustrated. Cloth, i.oo 

Muter. Practical and Analytical Chemistry. Fourth Edi- 
tion. Revised, to meet the requirements of American Medical 
Colleges, by Prof. C. C. Hamilton. Illustrated. Cloth, 2.00 

Holland. The Urine, Common Poisons, and Milk Analysis, 
Chemical and Microscopical. For Laboratory Use. Fourth 
Edition, Enlarged. Illustrated. Cloth, 1.00 

Van Niiys. Urine Analysis. Illus. Cloth, 2.00 

Wolff's Applied Medical Chemistry. By Lawrence Wolff, 
M.D., Dem. of Chemistry in Jefferson Medical College. Clo., i.oo 

CHILDREN. 

Goodhart and Starr. The Diseases of Children. Second 
Edition. By J. F. Goodhart, m.d.. Physician to the Evelina 
Hospital for Children; Assistant Physician to Guy's Hospital, 
London. Revised and Edited by Louis Starr, m.d., Clinical 
Professor of Diseases of Children in the Hospital of the Univer- 
sity of Pennsylvania; Physician to the Children's Hospital, 
Philadelphia. Containing many Prescriptions and Formulae, 
conforming to the U. S. Pharmacopoeia, Directions for making 
Artificial Human Milk, for the Artificial Digestion of Milk, etc. 
Illustrated. Cloth, 3.00; Leather, 3.50 

Hatfield. Diseases of Children. By M. P. Hatfield, m.d., 

Professor of Diseases of Children, Chicago Medical College. 

Colored Plate. i2mo. Cloth, i.oo; Interleaved, 1.25 

4^ See pages 14 and IS for list off Quiz- Compendsf 



8 STUDENTS' TEXT-BOOKS AND MANUALS. 

Children: — Continued. 
Starr. Diseases of the Digestive Organs in Infancy and 
Childhood. With chapters on the Investigation of Disease, 
and on the General Management of Children. By Louis Starr, 
M.D., Clinical Professor of Diseases of Children in the Univer- 
sity of Pennsylvania. lUus. Second Edition. Cloth, 2.25 

DENTISTRY. 

Fillebrown. Operative Dentistry. 330 Illus. Cloth, 2.50 

Flagg's Plastics and Plastic Filling. 4th Ed. Cloth, 4.00 
Gorgas. Dental Medicine. Fourth Edition. Cloth, 3.50 

Harris. Principles and Practice of Dentistry. Including 
Anatomy, Physiology, Pathology, Therapeutics, Dental Surgery 
and Mechanism. Twelfth Edition. Revised and enlarged by 
Professor Gorgas. 1028 Illustrations. Cloth, 7.00 ; Leather, 8.00 
Richardson's Mechanical Dentistry. Fifth Edition. 569 
Illustrations. 8vo. Cloth, 4.50; Leather, 5.50 

Sewill. Dental Surgery. 200 Illustrations. 3d Ed. Clo., 3.00 
Taft's Operative Dentistry. Dental Students and Practitioners. 
Fourth Edition. 100 Illustrations. Cloth, 4.25 ; Leather, 5.00 
Talbot. Irregularities of the Teeth, and their Treatment. 
Illustrated. 8vo. Second Edition. Cloth, 3.00 

Tomes' Dental Anatomy. Third Ed. 191 Illus. Cloth, 4.00 
Tomes' Dental Surgery. 3d Edition. 292 Illus. Cloth, 5.00 
Warren. Compend of Dental Pathology and Dental Medi- 
cine. Illustrated. Cloth, i.oo; Interleaved, 1.25 

DICTIONARIES. 

Gould's New Medical Dictionary. Containing the Definition 
and Pronunciation of all words in Medicine, with many useful 
Tables etc. J^ Dark Leather, 3.25 ; ^ Mor., Thumb Index, 4.25 

Harris' Dictionary of Dentistry. Fifth Edition. Completely 
revised by Prof. Gorgas. Cloth, 5.00; Leather, 6.00 

Cleaveland's Pronouncing Pocket Medical Lexicon. Small 
pocket size. Cloth, red edges .75 ; pocket-book style, i.oo 

Longley's Pocket Dictionary. The Student's Medical Lexicon, 
giving Definition and Pronunciation, with an Appendix giving 
Abbreviations used in Prescriptions, Metric Scale of Doses, etc. 
24mo. Cloth, I.oo; pocket-book Style, 1.25 

EYE. 

Hartridge on Refraction. 5th Edition. Illus. Cloth, 2.00 

Swanzy. Diseases of the Eye and their Treatment. 158 

Illustrations. Fourth Edition. Cloth, 3 00 

Fox and Gould. Compend of Diseases of the Eye and 

Refraction. 2d Ed. Enlarged. 71 Illus. 39 Formulae. 

Cloth, 1,00 ; Interleaved for Notes, 1.25 
4^ See^a^^es 2 to S /or list of Stiulents' Manuals, 



STUDENTS' TEXT-BOOKS AND MANUALS. 9 

ELECTRICITY. 

Bigelow. Plain Talks on Medical Electricity. Cloth, i.oo 

Mason's Compend of Medical Electricity. Cloth, z.oo 

Steavenson and Jones. Medical Electricity. A Practical 

Handbook. Just Ready. Illustrated. i2rao. Cloth, 2.50 

HYGIENE. 

Coplin's Practical Hygiene. By W. M. L. Coplin, Adjunct 
Professor of Hygiene, Jefferson Medical College, Philadelphia. 
Illustrated. In Press. 

Parkes' (Ed. A.) Practical Hygiene. Seventh Edition, en- 
larged. Illustrated. 8vo. Cloth, 4.50 

Parkes' (L. C.) Manual of Hygiene and Public Health. 
Second Edition. i2mo. Cloth, 2.50 

Wilson's Handbook of Hygiene and Sanitary Science. 
Seventh Edition. Revised and Illustrated. Cloth, 3.25 

MATERIA MEDICA AND THERAPEUTICS. 

Potter's Compend of Materia Medica, Therapeutics and 
Prescription Writing. Fifth Edition, revised and improved. 
See Page 75'. Cloth, i.oo; Interleaved for Notes, 1.25 

Biddle's Materia Medica. Eleventh Edition. By the late 
John B. Biddle, m.d. Revised by Clement Biddle, m.d., 8vo, 
illustrated. Cloth, 4.25; Leather, 5,00 

Potter. Handbook of Materia Medica, Pharmacy and 
Therapeutics. Including Action of Medicines, Special Thera- 
peutics, Pharmacology, etc. By Saml. O. L. Potter, m.d., 
M.R.c.p. (Lond.), Professor of the Practice of Medicine in 
Cooper Medical College, San Francisco. Third Revised and 
Enlarged Edition. 8vo. Cloth, 4.00; Leather, 5.00 

White and Wilcox. Materia Medica, Pharmacy, Phar- 
macology, and Therapeutics. A Handbook for Students. 
By Wm. Hale White, m.d., f.r.c.p., etc.. Physician to and 
Lecturer on Materia Medica, Guy's Hospital. Revised by 
Reynold W. Wilcox, m.d.. Professor of Clinical Medicine at the 
New York Post Graduate Medical School, Assistant Physician 
Bellevue Hospital, etc. American Edition. In Press. 

MEDICAL JURISPRUDENCE. 
Reese. A Text-book of Medical Jurisprudence and Toxi- 
cology. By John J. Reese, m.d.. Professor of Medical Juris- 
prudence and Toxicology in the Medical Department of the 
University of Pennsylvania ; President of the Medical Juris- 
prudence Society of Philadelphia ; Physician to St. Joseph's 
Hospital ; Corresponding Member of The New York Medico- 
legal Society, Third Edition. Cloth, 3.00; Leather, 3.50 
4^ See pages 14 and ij for list of f Quit- Compends ? 



10 STUDENTS' TEXT-BOOKS AND MANUALS. 

NERVOUS DISEASES. 

Gowers. Manual of Diseases of the Nervous System. 
A Complete Text-book. By William R. Gowers, m.d., Prof. 
Clinical Medicine, University College, London. Physician to 
National Hospital for the Paralyzed and Epileptic. Second 
Edition. Revised, Enlarged, and in many parts Rewritten. 
With many new Illustrations. Octavo. 

Vol. I. Diseases of the Nerves and Spinal Cord. 6i6 
pages. Cloth, 3.50 

Vol. II. Diseases of the Brain and Cranial Nerves. 
General and Functional Diseases. Nearly Ready. 

Ormerod. Diseases of Nervous System, Student's Guide to. 
By J. A. Ormerod, m.d., Oxon.,F.R.c.p. (London), Member Path- 
ological, Clinical, Ophthamological, and Neurological Societies, 
Physician to National Hospital for Paralyzed and Epileptic and 
to City of London Hospital for Diseases of the Chest, Demon- 
strator of Morbid Anatomy, St. Bartholomew's Hospital, etc. 
With 75 Wood Engravings. Cloth, 2.00 

OBSTETRICS AND GYNECOLOGY. 

Davis. A Manual of Obstetrics. By Edw. P. Davis, Dem- 
onstrator of Obstetrics, Jefferson Medical College, Philadelphia. 
Colored Plates, and 130 other Illustrations. i2mo. Cloth, 2.00 

Byford. Diseases of "Women. The Practice of Medicine and 
Surgery, as applied to the Diseases and Accidents Incident to 
Women. By W. H. Byford, a.m., m.d.. Professor of Gynaecology 
in Rush Medical College and of Obstetrics in the Woman's Med- 
ical College, etc., and Henry T. Byford, m.d.. Surgeon to the 
Woman's Hospital of Chicago. Fourth Edition. Revised and 
Enlarged. 306 Illustrations, over 100 of which are original. 
Octavo. 832 pages. Cloth, 5.00 ; Leather, 6.00 

Lewers' Diseases of V^omen, A Practical Text-book. 139 
Illustrations. Second Edition. Cloth, 2.50 

Parvin's Winckel's Diseases of Women. Second Edition. 

Including a Section on Diseases of the Bladder and Urethra. 
150 lUus. Revised. See j>age 3. Cloth, 3.00; Leather, 3.50 

Morris. Compend of Gynaecology. Illustrated. Cloth, i.oo 

WinckePs Obstetrics. A Text-book on Midwifery, includ- 
ing the Diseases of Childbed. By Dr. F. Winckel, Professor 
of Gynaecology, and Director of the Royal University Clinic for 
Women, in Munich. Authorized Translation, by J. Chfton 
Edgar, m.d.. Lecturer on Obstetrics, University Medical Col- 
lege, New York, with nearly 200 handsome Illustrations, the 
majority of which are original. 8vo. Cloth, 6.00; Leather, 7.00 

Landis' Compend of Obstetrics. Illustrated. 4th edition, 
enlarged. Cloth, i.oo; Interleaved lor Notes, 1.25 

Galabin*s Midwifery. By A. Lewis Galabin, m.d., f.r.c.p. 
227 Illustrations. See page 3. Cloth, 3.00; Leather, 3,50 

$f^ See pages 2 to sfor Ust of New Manuals^ 



STUDENTS' TEXT-BOOKS AND MANUALS. 



PATHOLOGY. HISTOLOGY. BIOLOGY. 

Bowlby. Surgical Pathology and Morbid Anatomy, for 
Students. 135 Illustrations. i2mo. Cloth, 2.00 

Davis' Elementary Biology. Illustrated. Cloth, 4.00 

Gilliam's Essentials of Pathology. A Handbook for Students. 
47 Illustrations. i2mo. Cloth, 2.00 

%* The object of this book is to unfold to the beginner the funda- 
mentals of pathology in a plain, practical way, and by bringing 
them within easy comprehension to increase his interest in the study 
of the subject. 

Gibbes' Practical Histology and Pathology. Third Edition. 
Enlarged, i2mo. Cloth, 1.75 

Virchow's Post-Mortem Examinations. 3d Ed. Cloth, i.oo 

PHYSICAL DIAGNOSIS. 

Fenwick. Student's Guide to Physical Diagnosis. 7th 

Edition. 117 Illustrations. i2mo. Cloth, 2.25 

Tyson's Student's Handbook of Physical Diagnosis. Illus- 
trated. i2mo. Cloth, 1.25 

PHYSIOLOGY. 

Yeo's Physiology. Fifth Edition. The most Popular Stu- 
dents' Book. By Gerald F. Yeo, m.d., f.r.c.s.. Professor of 
Physiology in King's College, London. Small Octavo. 758 
pages. 321 carefully printed Illustrations. With a Full 
Glossary and Index. See Page 3. Cloth, 3.00; Leather, 3.50 

Brubaker's Compend of Physiology. Illustrated. Sixth 
Edition. Cloth, i.oo; Interleaved for Notes, 1.25 

Stirling. Practical Physiology, including Chemical and Ex- 
perimental Physiology. 142 Illustrations. Cloth, 2.25 

Kirke's Physiology. New 12th Ed. Thoroughly Revised and 
Enlarged. 502 Illustrations. Cloth, 4.00 ; Leather, 5.00 

Landois' Human Physiology. Including Histology and Micro- 
scopical Anatomy, and with special reference to Practical Medi- 
cine. Fourth Edition. Translated and Edited by Prof. Stirling. 
845 Illustrations. Cloth, 7.00; Leather, 8.00 

'* With this Text-book at his command, no student could fail in 

his examination." — Lancets 

Sanderson's Physiological Laboratory. Being Practical Ex- 
ercises for the Student. 350 Illustrations. 8vo. Cloth, 5.00 

PRACTICE. 

Taylor. Practice of Medicine. A Manual. By Frederick 
Taylor, m.d.. Physician to, and Lecturer on Medicine at, Guy's 
Hospital, London ; Physician to Evelina Hospital for Sick Chil- 
dren, and Examiner in Materia Medica and Pharmaceutical 
Chemistry, University of London. Cloth, 4.00; Leather, 5.00 

4^ See pages 14 and IS for list of ? Quiz- Commends f 



12 STUDENTS' TEXT-BOOKS AND MANUALS. 

Practice : — Continued. 

Roberts* Practice. New Revised Edition. A Handbook 
of the Theory and Practice of Medicine. By Frederick T. 
Roberts, m.d., m.r.c.p., Professor of Clinical Medicine and 
Therapeutics in University College Hospital, London. Seventh 
Edition. Octavo. Cloth, 5.50; Sheep, 6,50 

Hughes. Compend of the Practice of Medicine. 4th Edi- 
tion. Two parts, each, Cloth, i.oo; Interleaved for Notes, 1.25 
Part i. — Continued, Eruptive and Periodical Fevers, Diseases 

of the Stomach, Intestines, Peritoneum, Biliary Passages, Liver, 

Kidneys, etc., and General Diseases, etc. 

Part ii. — Diseases of the Respiratory System, Circulatory 

System and Nervous System ; Diseases of the Blood, etc. 
Physicians' Edition. Fourth Edition. Including a Section 
on Skin Diseases. With Index, i vol. Full Morocco, Gilt, 2.50 

Frofn John A, Robinson, M.D., Assistant to Chair 0/ Clinical 
Medicine, now Lectiirer on Materia Medica, Rush Medical Col- 
lege, Chicago. 
*' Meets with my hearty approbation as a substitute for the 

ordinary note books almost universally used by medical students. 

It is concise, accurate, well arranged and lucid, . . . just the 

thing for students to use while studying physical diagnosis and the 

more practical departments of medicine." 

PRESCRIPTION BOOKS. 

Wythe's Dose and Symptom Book. Containing the Doses 
and Uses of all the principal Articles of the Materia Medica, etc. 
Seventeenth Edition. Completely Revised and Rewritten. Just 
Ready. 32mo. Cloth, i. 00; Pocket-book style, 1.25 

Pereira's Physician's Prescription Book. Containing Lists 
of Terms, Phrases, Contractions and Abbreviations used in 
Prescriptions Explanatory Notes, Grammatical Construction of 
Prescriptions, etc., etc. By Professor Jonathan Pereira, m.d. 
Sixteenth Edition. 32mo. Cloth, i. 00; Pocket-book style, 1.25 

PHARMACY. 

Ste^vart's Compend of Pharmacy. Based upon Remington's 
Text-book of Pharmacy. Third Edition, Revised. With new 
Tables, Index, Etc. Clothe i.oo ; Interleaved for Notes, 1.25 

Robinson. Latin Grammar of Pharmacy and Medicine. 
By H. D. Robinson, ph.d.. Professor of Latin Language and 
Literature, University of Kansas, Lawrence. With an Intro- 
duction by L. E. Sayre, ph.g., Professor of Pharmacy in, and 
Dean of, the Dept. of Pharmacy, University of Kansas. i2mo. 

Cloth, 2.00 

SKIN DISEASES. 

Anderson, (McCall) Skin Diseases. A complete Text-book, 
with Colored Plates and numerous Wood Engravings. 8vo. 

Cloth, 4.50; Leather, 5.50 

Van Harlingen on Skin Diseases. A Handbook of the Dis- 
eases of the Skin, their Diagnosis and Treatment (arranged alpha- 
betically). By Arthur Van Harlingen, m.d., Clinical Lecturer 
on Dermatology, Jefferson Medical College; Prof, of Diseases of 
the Skin in the Philadelphia Polyclinic. 2d Edition. Enlarged. 
With colored and other plates and illustrations. i2mo. Cloth, 2.50 
M^ See pages 2 to s for list of Neiv Manuals. 



STUDENTS' TEXT-BOOKS AND MANUALS. 13 

SURGERY AND BANDAGING. 

Moullin's Surgery. 500 Illustrations (some colored), 200 of 
which are original. Cloth, net 7.00; Leather, net 8.00 

Jacobson. Operations in Surgery. A Systematic Handbook 
for Physicians, Students and Hospital Surgeons. By W. H. A. 
Jacobson, b.a. Oxon., f.r.c.s. Eng. ; Ass't Surgeon Guy's Hos- 
pital ; Surgeon at Royal Hospital for Children and Women, etc. 
199 Illustrations. 1006 pages. . 8vo. Cloth. 5.00; Leather, 6.00 

Heath's Minor Surgery, and Bandaging. Ninth Edition. 142 
Illustrations. 60 Formulae and Diet Lists. Cloth, 2.00 

Horwitz's Compend of Surgery, Minor Surgery and 
Bandaging, Amputations, Fractures, Dislocations, Surgical 
Diseases, and the Latest Antiseptic Rules, etc., with Differential 
Diagnosis and Treatment. By Orville Horwitz, b.s., m.d., 
Demonstrator of Surgery, Jefferson Medical College. 4th edition. 
Enlarged and Rearranged. 136 Illustrations and 84 Formulae. 
i2mo. Cloth, I. GO ; Interleaved for the addition of Notes, 1.25 
***The new Section on Bandaging and Surgical Dressings, con- 
sists of 32 Pages and 41 Illustrations. Every Bandage of any 
importance is figured. This, with the Section on Ligation of 
Arteries, forms an ample Text-book for the Surgical Laboratory. 

Walsham. Manual of Practical Surgery. Third Edition. 
By Wm. J. Walsham, m.d., f.r.c.s., Asst. Surg, to, and Dem^ 
of Practical Surg, in, St. Bartholomew's Hospital; Surgeon to 
Metropolitan Free Hospital, London. With 318 Engravings. 
See Page 2. Cloth, 3.00; Leather, 3.50 

URINE, URINARY ORGANS, ETC. 

Holland. The Urine, and Common Poisons and The 
Milk. Chemical and Microscopical, for Laboratory Use. Illus- 
trated. Fourth Edition. i2mo. Interleaved. Cloth, 1.00 

Ralfe. Kidney Diseases and Urinary Derangements. 42 Illus- 
trations. i2mo. 572 pages. Cloth, 2.75 

Marshall and Smith. On the Urine. The Chemical Analysis ot 
the Urine. By John Marshall, m.d.. Chemical Laboratory, Univ. 
of Penna; and Prof. E. F. Smith, ph. d. Col. Plates. Cloth, i. 00 

Memminger. Diagnosis by the Urine. Illustrated. 

Cloth, 1. 00 

Tyson. On the Urine. A Practical Guide to the Examination 
of Urine. With Colored Plates and Wood Engravings. 7th YA. 
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Van Niiys, Urine Analysis. lUus. Cloth, 2.00 

VENEREAL DISEASES. 

Hill and Cooper. Student's Manual of Venereal Diseases, 
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4^ See pages 14 and i^ for list of f Quiz- Commends f 



NEW AND REVISED EDITIONS. 

PQUIZ-COMPENDS? 

The Best Compends for Students' Use 
in the Quiz Class, and when Pre- 
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Compiled in accordance with the latest teachings of promi- 
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They form a most complete, practical and exhaustive 
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with exceptional opportunities for noting the most recent 
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Cloth, each $i.oo. Interleaved for Notes, $1.25. 
No. I. HUMAN ANATOMY, '• Based upon Gray." Fifth 
Enlarged Edition, including Visceral Anatomy, formerly 
published separately. 16 Lithograph Plates, New 
Tables and 117 other Illustrations. By Samuel O. L. 
Potter, m.a., m.d., m.r.c.p. (Lond.), late A. A. Surgeon U. S. 
Army, Professor of Practice, Cooper Medical College, San Fran- 
cisco. 
Nos. 2 and 3. PRACTICE OF MEDICINE. Fourth Edi- 
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Medicine in Jefferson Medical College, Philadelphia. In two parts. 
Part I. — Continued, Eruptive and Periodical Fevers, Diseases 
of the Stomach, Intestines, Peritoneum, Biliary Passages, Liver, 
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*:}:* These little books can be regarded as a full set of notes upon 
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No. 4. PHYSIOLOGY, including Embryology. Sixth 
Edition. By Albert P. Brubaker, m.d.. Prof, of Physiology, 
Penn'a College of Dental Surgery ; Demonstrator of Physiology 
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with new Illustrations. 
No. 5. OBSTETRICS. Illustrated. Fourth Edition. By 
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Women, in Starling Medical College, Columbus, O. Revised 
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No. 6. MATERIA MEDICA, THERAPEUTICS AND 
PRESCRIPTION WRITING. Fifth Revised Edition. 
With especial Reference to the Physiological Action of Drugs, 
and a complete article on Prescription Writing. Based on the 
Last Revision of the U. S. Pharmacopoeia, and including many 
unofficinal remedies. By Samuel O. L. Potter, m.a., m.d., 
M.R.c.P. (Lond.), late A. A. Surg. U. S. Army ; Prof, of Practice, 
Cooper Medical College, San Francisco. Improved and Enlarged, 
with Index. 

No. 7. GYN/ECOLOGY. A Compend of Diseases of Women. 
By Henry Morris, m.d., Demonstrator of Obstetrics, Jefferson 
Medical College, Philadelphia. 45 Illustrations. 

No. 8. DISEASES OF THE EYE AND REFRACTION, 

including Treatment and Surgery. By L. Webster Fox, m.d.. 
Chief Clinical Assistant Ophthalmological Dept., Jefferson Med- 
ical College, etc., and Geo. M. Gould, m.d. 71 Illustrations, 39 
Formulae. Second Enlarged and Improved Edition. Index. 

No. 9, SURGERY, Minor Surgery and Bandaging. Illus- 
trated. Fourth Edition. Including Fractures, Wounds, 
Dislocations, Sprains, Amputations and other operations ; Inflam- 
mation, Suppuration, Ulcers, Syphilis, Tumors, Shock, etc. 
Diseases of the Spine, Ear, Bladder, Testicles, Anus, and 
other Surgical Diseases. By Orville Horwitz, a.m., m.d.. 
Demonstrator of Surgery, Jefferson Medical College. Revised 
and Enlarged. 84 Formulae and 136 Illustrations. 

No. 10. CHEMISTRY. Inorganic and Organic. For Medical 
and Dental Students. Including Urinary Analysis and Medical 
Chemistry. By Henry Leffmann, m.d., Prof, of Chemistry in 
Penn'a College of Dental Surgery, Phila. Third Edition, Revised 
and Rewritten, with Index. 

No. II. PHARMACY. Based upon *' Remington's Text-book 
of Pharmacy." By F. E. Stewart, m.d., ph. c, Quiz-Master 
at Philadelphia College of Pharmacy. Third Edition, Revised. 

No. 12. VETERINARY ANATOMY AND PHYSIOL- 
OGY. 29 Illustrations. By Wm. R. Ballou, m.d.. Prof, of 
Equine Anatomy at N. Y. College of Veterinary Surgeons. 

No. 13. DENTAL PATHOLOGY AND DENTAL MEDI- 
CINE. Containing all the most noteworthy points of interest 
to the Dental student. By Geo. W. Warren, d.d.s.. Clinical 
Chief, Penn'a College of Dental Surgery, Philadelphia. Illus. 

No. 14. DISEASES OF CHILDREN. By Dr. Marcus P. 
Hatfield, Prof, of Diseases of Children, Chicago Medical 
College. Colored Plate. 

Bouncl in Cloth, $1. Interleaved, for the Addition of Notes, $1.25. 



• These books are constantly revised to keep up with 
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Illustrated Descriptive Circular Free, 



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GOULD'S NEW 

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COMPACT. 
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UP TO DATE. 



It contains Tables of the Arteries, Bacilli, Gan- 
glia, Leucomaines, Micrococci, Muscles, 
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Small octavo, 520 pages, Half-Dark Leather, . ^3.25 
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**IJind it an excellent work, doing credit to the learning and 
discrimination of the author** 

*»* Sample Pag^es free. 






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